Expressed ligand—vascular intercellular signalling molecule

ABSTRACT

The present invention provides for a modified TIE-2 ligand which has been altered by addition, deletion or substitution of one or more amino acids, or by way of tagging, with for example, the Fc portion of human IgG-1, but which retains its ability to bind the TIE-2 receptor. The invention further provides for a modified TIE-2 ligand which is a chimeric TIE-2 ligand comprising at least a portion of a first TIE-2 ligand and a portion of a second TIE-2 ligand which is different from the first. In a specific embodiment, the invention further provides for a chimeric TIE ligand comprising at least a portion of TIE-2 Ligand-1 and a portion of TIE-2 Ligand-2. In addition the present invention provides for isolated nucleic acid molecule encoding the modified TIE-2 ligands described. The invention also provides for therapeutic compositions as well as a method of blocking blood vessel growth, a method of promoting neovascularization, a method of promoting the growth or differentiation of a cell expressing the TIE receptor, a method of blocking the growth or differentiation of a cell expressing the TIE receptor and a method of attenuating or preventing tumor growth in a human.

This application is a divisional application of U.S. Ser. No. 09/709,188filed 9 Nov. 2000, now U.S. Pat. No. 6,441,137, which is a continuationapplication of U.S. Ser. No. 08/740,223, filed on Oct. 25, 1996, nowU.S. Pat. No. 6,265,564, which claims the priority of U.S. Provisionalapplication No. 60/022,999 filed Aug. 2, 1996, now abandoned.

INTRODUCTION

The present invention relates generally to the field of geneticengineering and more particularly to genes for receptor tyrosine kinasesand their cognate ligands, their insertion into recombinant DNA vectors,and the production of the encoded proteins in recipient strains ofmicroorganisms and recipient eukaryotic cells. More specifically, thepresent invention is directed to a novel modified TIE-2 ligand thatbinds the TIE-2 receptor, as well as to methods of making and using themodified ligand. The invention further provides a nucleic acid sequenceencoding the modified ligand, and methods for the generation of nucleicacid encoding the modified ligand and the gene product. The modifiedTIE-2 ligand, as well as nucleic acid encoding it, may be useful in thediagnosis and treatment of certain diseases involving endothelial cellsand associated TIE receptors, such as neoplastic diseases involvingtumor angiogenesis, wound healing, thromboembolic diseases,atherosclerosis and inflammatory diseases. In addition, the modifiedligand may be used to promote the proliferation and/or differentiationof hematopoietic stem cells.

More generally, the receptor activating modified TIE-2 ligands describedherein may be used to promote the growth, survival, migration, and/ordifferentiation and/or stabilization or destabilization of cellsexpressing TIE receptor. Biologically active modified TIE-2 ligand maybe used for the in vitro maintenance of TIE receptor expressing cells inculture. Cells and tissues expressing TIE receptor include, for example,cardiac and vascular endothelial cells, lens epithelium and heartepicardium and early hematopoietic cells. Alternatively, such humanligand may be used to support cells which are engineered to express TIEreceptor. Further, modified TIE-2 ligand and its cognate receptor may beused in assay systems to identify further agonists or antagonists of thereceptor.

BACKGROUND OF THE INVENTION

The cellular behavior responsible for the development, maintenance, andrepair of differentiated cells and tissues is regulated, in large part,by intercellular signals conveyed via growth factors and similar ligandsand their receptors. The receptors are located on the cell surface ofresponding cells and they bind peptides or polypeptides known as growthfactors as well as other hormone-like ligands. The results of thisinteraction are rapid biochemical changes in the responding cells, aswell as a rapid and a long-term readjustment of cellular geneexpression. Several receptors associated with various cell surfaces maybind specific growth factors.

The phosphorylation of tyrosine residues in proteins by tyrosine kinasesis one of the key modes by which signals are transduced across theplasma membrane. Several currently known protein tyrosine kinase genesencode transmembrane receptors for polypeptide growth factors andhormones such as epidermal growth factor (EGF), insulin, insulin-likegrowth factor-I (IGF-I), platelet derived growth factors (PDGF-A and-B), and fibroblast growth factors (FGFs). (Heldin et al., CellRegulation, 1: 555-566 (1990); Ullrich, et al., Cell, 61: 243-54(1990)). In each instance, these growth factors exert their action bybinding to the extracellular portion of their cognate receptors, whichleads to activation of the intrinsic tyrosine kinase present on thecytoplasmic portion of the receptor. Growth factor receptors ofendothelial cells are of particular interest due to the possibleinvolvement of growth factors in several important physiological andpathological processes, such as vasculogenesis, angiogenesis,atherosclerosis, and inflammatory diseases. (Folkman, et al. Science,235: 442-447 (1987)). Also, the receptors of several hematopoieticgrowth factors are tyrosine kinases; these include c-fms, which is thecolony stimulating factor 1 receptor, Sherr, et al., Cell, 41: 665-676(1985), and c-kit, a primitive hematopoietic growth factor receptorreported in Huang, et al., Cell, 63: 225-33 (1990).

The receptor tyrosine kinases have been divided into evolutionarysubfamilies based on the characteristic structure of their ectodomains.(Ullrich, et al. Cell, 61: 243-54 (1990)). Such subfamilies include, EGFreceptor-like kinase (subclass I) and insulin receptor-like kinase(subclass II), each of which contains repeated homologous cysteine-richsequences in their extracellular domains. A single cysteine-rich regionis also found in the extracellular domains of the eph-like kinases.Hirai, et al., Science, 238: 1717-1720 (1987); Lindberg, et al. Mol.Cell. Biol., 10: 6316-24 (1990); Lhotak, et al., Mol. Cell. Biol. 11:2496-2502 (1991). PDGF receptors as well as c-fms and c-kit receptortyrosine kinases may be grouped into subclass III; while the FGFreceptors form subclass IV. Typical for the members of both of thesesubclasses are extracellular folding units stabilized by intrachaindisulfide bonds. These so-called immunoglobulin (Ig)-like folds arefound in the proteins of the immunoglobulin superfamily which contains awide variety of other cell surface receptors having either cell-bound orsoluble ligands. Williams, et al., Ann. Rev. Immunol., 6: 381-405(1988).

Receptor tyrosine kinases differ in their specificity and affinity. Ingeneral, receptor tyrosine kinases are glycoproteins which consist of(1) an extracellular domain capable of binding the specific growthfactor(s); (2) a transmembrane domain which usually is an alpha-helicalportion of the protein; (3) a juxtamembrane domain where the receptormay be regulated by, e.g., protein phosphorylation; (4) a tyrosinekinase domain which is the enzymatic component of the receptor; and (5)a carboxyterminal tail which in many receptors is involved inrecognition and binding of the substrates for the tyrosine kinase.

Processes such as alternative exon splicing and alternative choice ofgene promoter or polyadenylation sites have been reported to be capableof producing several distinct polypeptides from the same gene. Thesepolypeptides may or may not contain the various domains listed above. Asa consequence, some extracellular domains may be expressed as separate,secreted proteins and some forms of the receptors may lack the tyrosinekinase domain and contain only the extracellular domain inserted in theplasma membrane via the transmembrane domain plus a short carboxylterminal tail.

A gene encoding an endothelial cell transmembrane tyrosine kinase,originally identified by RT-PCR as an unknown tyrosine kinase-homologouscDNA fragment from human leukemia cells, was described by Partanen, etal., Proc. Natl. Acad. Sci. USA, 87: 8913-8917 (1990). This gene and itsencoded protein are called “TIE” which is an abbreviation for “tyrosinekinase with Ig and EGF homology domains.” Partanen, et al. Mol. Cell.Biol. 12: 1698-1707 (1992).

It has been reported that tie mRNA is present in all human fetal andmouse embryonic tissues. Upon inspection, tie message has been localizedto the cardiac and vascular endothelial cells. Specifically, tie mRNAhas been localized to the endothelia of blood vessels and endocardium of9.5 to 18.5 day old mouse embryos. Enhanced tie expression was shownduring neovascularization associated with developing ovarian folliclesand granulation tissue in skin wounds. Korhonen, et al. Blood 80:2548-2555 (1992). Thus the TIEs have been suggested to play a role inangiogenesis, which is important for developing treatments for solidtumors and several other angiogenesis-dependent diseases such asdiabetic retinopathy, psoriasis, atherosclerosis and arthritis.

Two structurally related rat TIE receptor proteins have been reported tobe encoded by distinct genes with related profiles of expression. Onegene, termed tie-1, is the rat homolog of human tie. Maisonpierre, etal., Oncogene 8: 1631-1637 (1993). The other gene, tie-2, may be the rathomolog of the murine tek gene, which, like tie, has been reported to beexpressed in the mouse exclusively in endothelial cells and theirpresumptive progenitors. Dumont, et al. Oncogene 8: 1293-1301 (1993).The human homolog of tie-2 is described in Ziegler, U.S. Pat. No.5,447,860 which issued on Sep. 5, 1995 (wherein it is referred to as“ork”), which is incorporated in its entirety herein.

Both genes were found to be widely expressed in endothelial cells ofembryonic and postnatal tissues. Significant levels of tie-2 transcriptswere also present in other embryonic cell populations, including lensepithelium, heart epicardium and regions of mesenchyme. Maisonpierre, etal., Oncogene 8: 1631-1637 (1993).

The predominant expression of the TIE receptor in vascular endotheliasuggests that TIE plays a role in the development and maintenance of thevascular system. This could include roles in endothelial celldetermination, proliferation, differentiation and cell migration andpatterning into vascular elements. Analyses of mouse embryos deficientin TIE-2 illustrate its importance in angiogenesis, particularly forvascular network formation in endothelial cells. Sato, T. N., et al.,Nature 376:70-74 (1995). In the mature vascular system, the TIEs couldfunction in endothelial cell survival, maintenance and response topathogenic influences.

The TIE receptors are also expressed in primitive hematopoietic stemcells, B cells and a subset of megakaryocytic cells, thus suggesting therole of ligands which bind these receptors in early hematopoiesis, inthe differentiation and/or proliferation of B cells, and in themegakaryocytic differentiation pathway. Iwama, et al. Biochem. Biophys.Research Communications 195:301-309 (1993); Hashiyama, et al. Blood87:93-101 (1996), Batard, et al. Blood 87:2212-2220 (1996).

SUMMARY OF THE INVENTION

The present invention provides for a composition comprising a modifiedTIE-2 ligand substantially free of other proteins. As used herein,modified TIE-2 ligand refers to a ligand of the TIE family of ligands,whose representatives comprise ligands TL1, TL2, TL3 and TL4 asdescribed herein, which has been altered by addition, deletion orsubstitution of one or more amino acids, or by way of tagging, with forexample, the Fc portion of human IgG-1, but which retains its ability tobind the TIE-2 receptor. Modified TIE-2 ligand also includes a chimericTIE-2 ligand comprising at least a portion of a first TIE-2 ligand and aportion of a second TIE-2 ligand which is different from the first. Byway of non-limiting example, the first TIE-2 ligand is TL1 and thesecond TIE-2 ligand is TL2. The invention envisions other combinationsusing additional TIE-2 ligand family members. For example, othercombinations for creating a chimeric TIE-2 ligand are possible,including but not limited to those combinations wherein the first ligandis selected from the group consisting of TL1, TL2, TL3 and TL4, and thesecond ligand, different from the first ligand, is selected from thegroup consisting of TL1, TL2, TL3 and TL4.

The invention also provides for an isolated nucleic acid moleculeencoding a modified TIE-2 ligand. In one embodiment, the isolatednucleic acid molecule encodes a TIE-2 ligand of the TIE family ofligands, whose representatives comprise ligands TL1, TL2, TL3 and TL4 asdescribed herein, which has been altered by addition, deletion orsubstitution of one or more amino acids, or by way of tagging, with forexample, the Fc portion of human IgG-1, but which retains its ability tobind the TIE-2 receptor. In another embodiment, the isolated nucleicacid molecule encodes a modified TIE-2 ligand which is a chimeric TIE-2ligand comprising at least a portion of a first TIE-2 ligand and aportion of a second TIE-2 ligand which is different from the first. Byway of non-limiting example, the first TIE-2 ligand is TL1 and thesecond TIE-2 ligand is TL2. The invention envisions other combinationsusing additional TIE-2 ligand family members. For example, othercombinations are possible, including but not limited to thosecombinations wherein the isolated nucleic acid molecule encodes amodified TIE-2 ligand which is a chimeric TIE-2 ligand comprising aportion of a first ligand selected from the group consisting of TL1,TL2, TL3 and TL4, and a portion of a second ligand, different from thefirst ligand, selected from the group consisting of TL1, TL2, TL3 andTL4.

The isolated nucleic acid may be DNA, cDNA or RNA. The invention alsoprovides for a vector comprising an isolated nucleic acid moleculeencoding a modified TIE-2 ligand. The invention further provides for ahost-vector system for the production in a suitable host cell of apolypeptide having the biological activity of a modified TIE-2 ligand.The suitable host cell may be bacterial, yeast, insect or mammalian. Theinvention also provides for a method of producing a polypeptide havingthe biological activity of a modified TIE-2 ligand which comprisesgrowing cells of the host-vector system under conditions permittingproduction of the polypeptide and recovering the polypeptide soproduced.

The invention herein described of an isolated nucleic acid moleculeencoding a modified TIE-2 ligand further provides for the development ofthe ligand as a therapeutic for the treatment of patients suffering fromdisorders involving cells, tissues or organs which express the TIE-2receptor. The present invention also provides for an antibody whichspecifically binds such a therapeutic molecule. The antibody may bemonoclonal or polyclonal. The invention also provides for a method ofusing such a monoclonal or polyclonal antibody to measure the amount ofthe therapeutic molecule in a sample taken from a patient for purposesof monitoring the course of therapy.

The present invention also provides for an antibody which specificallybinds a modified TIE-2 ligand as described herein. The antibody may bemonoclonal or polyclonal. Thus the invention further provides fortherapeutic compositions comprising an antibody which specifically bindsa modified TIE-2 ligand, in a pharmaceutically acceptable vehicle. Theinvention also provides for a method of blocking blood vessel growth ina mammal by administering an effective amount of a therapeuticcomposition comprising an antibody which specifically binds a receptoractivating modified TIE-2 ligand as described herein, in apharmaceutically acceptable vehicle.

The invention further provides for therapeutic compositions comprising amodified TIE-2 ligand as described herein, in a pharmaceuticallyacceptable vehicle. The invention also provides for a method ofpromoting neovascularization in a patient by administering an effectiveamount of a therapeutic composition comprising a receptor activatingmodified TIE-2 ligand as described herein, in a pharmaceuticallyacceptable vehicle. In one embodiment, the method may be used to promotewound healing. In another embodiment, the method may be used to treatischemia. In yet another embodiment, a receptor activating modifiedTIE-2 ligand as described herein is used, alone or in combination withother hematopoietic factors, to promote the proliferation ordifferentiation of hematopoietic stem cells, B cells or megakaryocyticcells.

Alternatively, the invention provides that a modified TIE-2 ligand maybe conjugated to a cytotoxic agent and a therapeutic compositionprepared therefrom. The invention further provides for a receptorbodywhich specifically binds a modified TIE-2 ligand. The invention furtherprovides for therapeutic compositions comprising a receptorbody whichspecifically binds a modified TIE-2 ligand in a pharmaceuticallyacceptable vehicle. The invention also provides for a method of blockingblood vessel growth in a mammal by administering an effective amount ofa therapeutic composition comprising a receptorbody which specificallybinds a modified TIE-2 ligand in a pharmaceutically acceptable vehicle.

The invention also provides for a TIE-2 receptor antagonist as well as amethod of inhibiting TIE-2 biological activity in a mammal comprisingadministering to the mammal an effective amount of a TIE-2 antagonist.According to the invention, the antagonist may be a modified TIE-2ligand as described herein which binds to, but does not activate, theTIE-2 receptor.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B—TIE-2 receptorbody (TIE-2 RB) inhibits the developmentof blood vessels in the embryonic chicken chorioallantoic membrane(CAM). A single piece of resorbable gelatin foam (Gelfoam) soaked with 6μg of RB was inserted immediately under the CAM of 1-day chick embryos.After 3 further days of incubation, 4 day old embryos and surroundingCAM were removed and examined. FIG. 1A: embryos treated with EHK-1 RB(rEHK-1 ecto/hlgG1 Fc) were viable and possessed normally developedblood vessels in their surrounding CAM. FIG. 1B: all embryos treatedwith TIE-2 RB (r TIE-2 ecto/hlgG1 Fc) were dead, diminished in size andwere almost completely devoid of surrounding blood vessels.

FIG. 2—Vector pJFE14.

FIG. 3—Restriction map of λgt10.

FIGS. 4A-4D—Nucleic acid (SEQ ID NO: 1) and deduced amino acid (SEQ IDNO: 2) (single letter code) sequences of human TIE-2 ligand 1 from cloneλgt10 encoding htie-2 ligand 1.

FIGS. 5A-5D—Nucleic acid (SEQ ID NO: 3) and deduced amino acid (SEQ IDNO: 4) (single letter code) sequences of human TIE-2 ligand 1 from T98Gclone.

FIGS. 6A-6D—Nucleic acid and deduced amino acid (single letter code)sequences of human TIE-2 ligand 2 from clone pBluescript KS encodinghuman TIE 2 ligand 2.

FIG. 7—Western blot showing activation of TIE-2 receptor by TIE-2 ligand1 (Lane L1) but not by TIE-2 ligand 2 (Lane L2) or control (Mock).

FIG. 8—Western blot showing that prior treatment of HAEC cells withexcess TIE-2 ligand 2 (Lane 2) antagonizes the subsequent ability ofdilute TIE-2 ligand 1 to activate the TIE-2 receptor (TIE2-R) ascompared with prior treatment of HAEC cells with MOCK medium (Lane 1).

FIG. 9—Western blot demonstrating the ability of TL2 to competitivelyinhibit TL1 activation of the TIE-2 receptor using the human cell hybridline, EA.hy926.

FIGS. 10A-10D—Histogram representation of binding to rat TIE-2 IgGimmobilized surface by TIE-2 ligand in C2C12 ras (FIG. 10A), Rat2 ras(FIG. 10B), SHEP (FIG. 10C), and T98G (FIG. 10D) concentrated (10×)conditioned medium. Rat TIE-2 (rTIE2) specific binding is demonstratedby the significant reduction in the binding activity in the presence of25 μg/ml soluble rat TIE-2 RB as compared to a minor reduction in thepresence of soluble trkB RB.

FIGS. 11A-11B—Binding of recombinant human TIE-2 ligand 1 (hTL1) (FIG.11A) and human TIE-2 ligand 2 (hTL2) (FIG. 11B), in COS cellsupernatants, to a human TIE-2 receptorbody (RB) immobilized surface.Human TIE-2-specific binding was determined by incubating the sampleswith 25 μg/ml of either soluble human TIE-2 RB or trkB RB; significantreduction in the binding activity is observed only for the samplesincubated with human TIE-2 RB.

FIG. 12—Western blot showing that TIE-2 receptorbody (denoted TIE-2 RBor, as here, TIE2-Fc) blocks the activation of TIE-2 receptors by TIE-2ligand 1 (TL1) in HUVEC cells, whereas an unrelated receptorbody(TRKB-Fc) does not block this activation.

FIG. 13—Agarose gels showing serial dilutions [undiluted (1) to 10⁻⁴] ofthe TL1 and TL2 RT-PCR products obtained from E14.5 mouse fetal liver(Lanes 1-total, Lanes 3-stromal enriched, and Lanes 4—c-kit⁺TER119hematopoietic precursor cells) and E14.5 mouse fetal thymus (Lanes2-total).

FIG. 14—Agarose gels showing serial dilutions [undiluted (1) to 10⁻³] ofthe TL1 and TL2 RT-PCR products obtained from E17.5 mouse fetal thymuscortical stromal cells (Lanes 1-CDR1+/A2B5−) and medullary stromal cells(Lane CDR1−/A2B5+).

FIG. 15—A schematic representation of the hypothesized role of theTIE-2/TIE ligands in angiogenesis. TL1 is represented by (•), TL2 isrepresented by (*), TIE-2 is represented by (T), VEGF is represented by([ ]), and flk-1 (a VEGF receptor) is represented by (Y).

FIG. 16—In situ hybridization slides showing the temporal expressionpattern of TIE-2, TL1, TL2, and VEGF during angiogenesis associated withfollicular development and corpus luteum formation in the ovary of a ratthat was treated with pregnant mare serum. Column 1: Early pre-ovulatoryfollicle; Column 2: pre-ovulatory follicle; Column 3: early corpusluteum; and Column 4: atretic follicle; Row A: bright field; Row B:VEGF; Row C: TL2; Row D: TL1 and Row E: TIE-2 receptor.

FIG. 17—Comparison of amino acid sequences of mature TL1 protein (SEQ IDNO: 7) and mature TL2 protein (SEQ ID NO: 8). The TL1 sequence is thesame as that set forth in FIGS. 4A-4D (SEQ ID NO: 1 AND SEQ ID NO: 2),except that the putative leader sequence has been removed. Similarly,the TL2 sequence is the same as that set forth in FIGS. 6A-6D (SEQ IDNO: 5 and SEQ ID NO: 6), except that the putative leader sequence hasbeen removed. Arrows indicate residues Arg49, Cys245 and Arg264 of TL1,which correspond to the residues at amino acid positions 69, 265 and284, respectively, of TL1 as set forth in FIGS. 4A-4D (SEQ ID NO: 1 andSEQ ID NO: 2).

FIG. 18—Western blot of the covalent multimeric structure of TL1 and TL2(Panel A) and the interconversion of TL1 and TL2 by the mutation of onecysteine (Panel B).

FIG. 19—A typical curve of TIE-2-IgG binding to immobilized TL1 in aquantitative cell-free binding assay.

FIG. 20—A typical curve showing TIE-2 ligand 1 ligandbody comprising thefibrinogen-like domain of the ligand bound to the Fc domain of IgG(TL1-fFc) binding to immobilized TIE-2 ectodomain in a quantitativecell-free binding assay.

FIGS. 21A-21C—Nucleotide (SEQ ID NO: 9) and deduced amino acid (SEQ IDNO: 10) (single letter code) sequences of TIE ligand-3. The codingsequence starts at position 47. The fibrinogen-like domain starts atposition 929.

FIGS. 22A-22B—Comparison of Amino Acid Sequences of TIE Ligand FamilyMembers. mTL3=mouse TIE ligand-3 (SEQ ID NO: 11); hTL1=human TIE-2ligand1 (SEQ ID NO: 12); chTL1=chicken TIE-2 ligand1 (SEQ ID NO; 13);mTL1=mouse TIE-2 ligand 1 (SEQ ID NO: 14); mTL2=mouse TIE-2 ligand 2(SEQ ID NO: 15); hTL2=human TIE-2 ligand 2 (SEQ ID NO: 16). The boxedregions indicate conserved regions of homology among the family members.

FIGS. 23A-23C—Nucleotide (SEQ ID NO: 17) and deduced amino acid (SEQ IDNO: 18) (single letter code) sequences of TIE ligand-4. Arrow indicatesnucleotide position 569.

FIGS. 24A-24C—Nucleotide (SEQ ID NO: 19) and deduced amino acid (SEQ IDNO: 20) (single letter code) sequences of chimeric TIE ligand designated1N1C2F (chimera 1). The putative leader sequence is encoded bynucleotides 1-60.

FIGS. 25A-25C—Nucleotide (SEQ ID NO: 21) and deduced amino acid (SEQ IDNO: 22) (single letter code) sequences of chimeric TIE ligand designated2N2C1F (chimera 2). The putative leader sequence is encoded bynucleotides 1-48.

FIGS. 26A-26C—Nucleotide (SEQ ID NO: 23) and deduced amino acid (SEQ IDNO: 24) (single letter code) sequences of chimeric TIE ligand designated1N2C2F (chimera 3). The putative leader sequence is encoded bynucleotides 1-60.

FIGS. 27A-27C—Nucleotide (SEQ ID NO: 25) and deduced amino acid (SEQ IDNO: 26) (single letter code) sequences of chimeric TIE ligand designated2N1C1F (chimera 4). The putative leader sequence is encoded bynucleotides 1-48.

DETAILED DESCRIPTION OF THE INVENTION

As described in greater detail below, applicants have created novelmodified TIE-2 ligands that bind the TIE-2 receptor. The presentinvention provides for a composition comprising a modified TIE-2 ligandsubstantially free of other proteins. As used herein, modified TIE-2ligand refers to a ligand of the TIE family of ligands, whoserepresentatives comprise ligands TL1, TL2, TL3 and TL4 as describedherein, which has been altered by addition, deletion or substitution ofone or more amino acids, or by way of tagging, with for example, the Fcportion of human IgG-1, but which retains its ability to bind the TIE-2receptor. Modified TIE-2 ligand also includes a chimeric TIE-2 ligandcomprising at least a portion of a first TIE-2 ligand and a portion of asecond TIE-2 ligand which is different from the first. By way ofnon-limiting example, the first TIE-2 ligand is TL1 and the second TIE-2ligand is TL2. The invention envisions other combinations usingadditional TIE-2 ligand family members. For example, other combinationsfor creating a chimeric TIE-2 ligand are possible,)including but notlimited to those combinations wherein the first ligand is selected fromthe group consisting of TL1, TL2, TL3 and TL4, and the second ligand,different from the first ligand, is selected from the group consistingof TL1, TL2, TL3 and TL4.

The invention also provides for an isolated nucleic acid moleculeencoding a modified TIE-2 ligand. In one embodiment, the isolatednucleic acid molecule encodes a TIE-2 ligand of the TIE family ofligands, whose representatives comprise ligands TL1, TL2, TL3 and TL4 asdescribed herein, which has been altered by addition, deletion orsubstitution of one or more amino acids, or by way of tagging, with forexample, the Fc portion of human IgG-1, but which retains its ability tobind the TIE-2 receptor. In another embodiment, the isolated nucleicacid molecule encodes a modified TIE-2 ligand which is a chimeric TIE-2ligand comprising at least a portion of a first TIE-2 ligand and aportion of a second TIE-2 ligand which is different from the first. Byway of non-limiting example, the first TIE-2 ligand is TL1 and thesecond TIE-2 ligand is TL2. The invention envisions other combinationsusing additional TIE-2 ligand family members. For example, othercombinations are possible, including but not limited to thosecombinations wherein the isolated nucleic acid molecule encodes amodified TIE-2 ligand which is a chimeric TIE-2 ligand comprising aportion of a first ligand selected from the group consisting of TL1,TL2, TL3 and TL4, and a portion of a second ligand, different from thefirst ligand, selected from the group consisting of TL1, TL2, TL3 andTL4.

The present invention comprises the modified TIE-2 ligands and theiramino acid sequences, as well as functionally equivalent variantsthereof, as well as proteins or peptides comprising substitutions,deletions or insertional mutants of the described sequences, which bindTIE-2 receptor and act as agonists or antagonists thereof. Such variantsinclude those in which amino acid residues are substituted for residueswithin the sequence resulting in a silent change. For example, one ormore amino acid residues within the sequence can be substituted byanother amino acid(s) of a similar polarity which acts as a functionalequivalent, resulting in a silent alteration. Substitutes for an aminoacid within the sequence may be selected from other members of the classto which the amino acid belongs. For example, the class of nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan and methionine. The polar neutralamino acids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine. The positively charged (basic) amino acidsinclude arginine, lysine and histidine. The negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

Also included within the scope of the invention are proteins orfragments or derivatives thereof which exhibit the same or similarbiological activity as the modified TIE-2 ligands described herein, andderivatives which are differentially modified during or aftertranslation, e.g., by glycosylation, proteolytic cleavage, linkage to anantibody molecule or other cellular ligand, etc. Functionally equivalentmolecules also include molecules that contain modifications, includingN-terminal modifications, which result from expression in a particularrecombinant host, such as, for example, N-terminal methylation whichoccurs in certain bacterial (e.g. E. coli) expression systems.

The present invention also encompasses the nucleotide sequences thatencode the proteins described herein as modified TIE-2 ligands, as wellas host cells, including yeast, bacteria, viruses, and mammalian cells,which are genetically engineered to produce the proteins, by e.g.transfection, transduction, infection, electroporation, ormicroinjection of nucleic acid encoding the modified TIE-2 ligandsdescribed herein in a suitable expression vector. The present inventionalso encompasses introduction of the nucleic acid encoding modifiedTIE-2 ligands through gene therapy techniques such as is described, forexample, in Finkel and Epstein FASEB J. 9:843-851 (1995); Guzman, et al.PNAS (USA) 91:10732-10736 (1994).

One skilled in the art will also recognize that the present inventionencompasses DNA and RNA sequences that hybridize to a modified TIE-2ligand encoding nucleotide sequence, under conditions of moderatestringency, as defined in, for example, Sambrook, et al. MolecularCloning: A Laboratory Manual, 2 ed. Vol. 1, pp. 101-104, Cold SpringHarbor Laboratory Press (1989). Thus, a nucleic acid moleculecontemplated by the invention includes one having a nucleotide sequencededuced from an amino acid sequence of a modified TIE-2 ligand preparedas described herein, as well as a molecule having a sequence ofnucleotides that hybridizes to such a nucleotide sequence, and also anucleotide sequence which is degenerate of the above sequences as aresult of the genetic code, but which encodes a ligand that binds TIE-2receptor and which has an amino acid sequence and other primary,secondary and tertiary characteristics that are sufficiently duplicativeof a modified TIE-2 ligand described herein so as to confer on themolecule the same biological activity as the modified TIE-2 liganddescribed herein.

The present invention provides for an isolated nucleic acid moleculeencoding a modified TIE-2 ligand that binds and activates TIE-2 receptorcomprising a nucleotide sequence encoding TIE-2 ligand 1 wherein theportion of the nucleotide sequence that encodes the N-terminal domain ofTIE-2 ligand 1 is replaced by a nucleotide sequence that encodes theN-terminal domain of TIE-2 ligand 2. The invention also provides forsuch a nucleic acid molecule, with a further modification such that theportion of the nucleotide sequence that encodes the coiled-coil domainof TIE-2 ligand 1 is replaced by a nucleotide sequence that encodes thecoiled-coil domain of TIE-2 ligand 2.

The present invention also provides for an isolated nucleic acidmolecule encoding a modified TIE-2 ligand that binds and activates TIE-2receptor comprising a nucleotide sequence encoding TIE-2 ligand 1wherein the portion of the nucleotide sequence that encodes theN-terminal domain of TIE-2 ligand 1 is replaced by a nucleotide sequencethat encodes the N-terminal domain of TIE-2 ligand 2 and which isfurther modified to encode a different amino acid instead of thecysteine residue encoded by nucleotides 784-786 as set forth in FIGS.27A-27C (SEQ ID NO: 25 and SEQ ID NO: 26). A serine residue ispreferably substituted for the cysteine residue. In another embodiment,the nucleic acid molecule is further modified to encode a differentamino acid instead of the arginine residue encoded by nucleotides199-201 as set forth in FIGS. 27A-27C (SEQ ID NO: 25 and SEQ ID NO: 26).A serine residue is preferably substituted for the arginine residue.

The present invention also provides for an isolated nucleic acidmolecule encoding a modified TIE-2 ligand that binds and activates TIE-2receptor comprising a nucleotide sequence encoding TIE-2 ligand 1 whichis modified to encode a different amino acid instead of the cysteineresidue at amino acid position 245. A serine residue is preferablysubstituted for the cysteine residue.

The invention further provides for an isolated nucleic acid moleculeencoding a modified TIE-2 ligand that binds but does not activate TIE-2receptor comprising a nucleotide sequence encoding TIE-2 ligand 1wherein the portion of the nucleotide sequence that encodes theN-terminal domain of TIE-2 ligand 1 is deleted. The invention alsoprovides for such a nucleic acid molecule further modified so that theportion of the nucleotide sequence that encodes the coiled-coil domainof TIE-2 ligand 1 is deleted and the portion encoding thefibrinogen-like domain is fused in-frame to a nucleotide sequenceencoding a human immunoglobulin gamma-1 constant region (IgG1 Fc).

The invention further provides for an isolated nucleic acid moleculeencoding a modified TIE-2 ligand that binds but does not activate TIE-2receptor comprising a nucleotide sequence encoding TIE-2 ligand 2wherein the portion of the nucleotide sequence that encodes theN-terminal domain of TIE-2 ligand 2 is deleted. The invention alsoprovides for such a nucleic acid molecule further modified so that theportion of the nucleotide sequence that encodes the coiled-coil domainof TIE-2 ligand 2 is deleted and the portion encoding thefibrinogen-like domain is fused in-frame to a nucleotide sequenceencoding a human immunoglobulin gamma-1 constant region (IgG1 Fc).

The invention further provides for an isolated nucleic acid moleculeencoding a modified TIE-2 ligand that binds but does not activate TIE-2receptor comprising a nucleotide sequence encoding TIE-2 ligand 1wherein the portion of the nucleotide sequence that encodes thefibrinogen-like domain of TIE-2 ligand 1 is replaced by a nucleotidesequence that encodes the fibrinogen-like domain of TIE-2 ligand 2. Theinvention also provides for such a nucleic acid molecule furthermodified so that the portion of the nucleotide sequence that encodes thecoiled-coil domain of TIE-2 ligand 1 is replaced by a nucleotidesequence that encodes the coiled-coil domain of TIE-2 ligand 2.

The invention further provides for a modified TIE-2 ligand encoded byany of nucleic acid molecules of the invention.

The present invention also provides for a chimeric TIE-2 ligandcomprising at least a portion of a first TIE-2 ligand and a portion of asecond TIE-2 ligand which is different from the first, wherein the firstand second TIE-2 ligands are selected from the group consisting of TIE-2Ligand-1, TIE-2 Ligand-2, TIE Ligand-3 and TIE Ligand-4. Preferably, thechimeric TIE ligand comprises at least a portion of TIE-2 Ligand-1 and aportion of TIE-2 Ligand-2.

The invention also provides a nucleic acid molecule that encodes achimeric TIE ligand as set forth in FIGS. 24A-24C (SEQ ID NO: 19 and SEQID NO: 20), 25A-25C (SEQ ID NO: 21 and SEQ ID NO: 22), 26A-26C (SEQ IDNO: 23 and SEQ ID NO: 24), or 27A-27C (SEQ ID NO: 25 and (SEQ ID NO:26). The invention also provides a chimeric TIE ligand as set forth inFIGS. 24A-24C (SEQ ID NO: 19 and SEQ ID NO: 20), 25A-25C (SEQ ID NO: 21and SEQ ID NO: 22), 26A-26C (SEQ ID NO: 23 and SEQ ID NO: 24), or27A-27C (SEQ ID NO: 25 and SEQ ID NO: 26). The invention furtherprovides a chimeric TIE ligand as set forth in FIGS. 27A-27C (SEQ ID NO:25 and SEQ ID NO: 26), modified to have a different amino acid insteadof the cysteine residue encoded by nucleotides 784-786.

Any of the methods known to one skilled in the art for the insertion ofDNA fragments into a vector may be used to construct expression vectorsencoding a modified TIE-2 ligand using appropriatetranscriptional/translational control signals and the protein codingsequences. These methods may include in vitro recombinant DNA andsynthetic techniques and in vivo recombinations (genetic recombination).Expression of a nucleic acid sequence encoding a modified TIE-2 ligandor peptide fragments thereof may be regulated by a second nucleic acidsequence which is operably linked to the a modified TIE-2 ligandencoding sequence such that the modified TIE-2 ligand protein or peptideis expressed in a host transformed with the recombinant DNA molecule.For example, expression of a modified TIE-2 ligand described herein maybe controlled by any promoter/enhancer element known in the art.Promoters which may be used to control expression of the ligand include,but are not limited to the long terminal repeat as described in Squintoet al., (Cell 65:1-20 (1991)); the SV40 early promoter region (Bernoistand Chambon, Nature 290:304-310), the CMV promoter, the M-MuLV 5′terminal repeat, the promoter contained in the 3′ long terminal repeatof Rous sarcoma virus (Yamamoto, et al., Cell 22:787-797 (1980)), theherpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci.U.S.A. 78:144-1445 (1981)), the adenovirus promoter, the regulatorysequences of the metallothionein gene (Brinster et al., Nature 296:39-42(1982)); prokaryotic expression vectors such as the β-lactamase promoter(Villa-Kamaroff, et al., Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731(1978)), or the tac promoter (DeBoer, et al., Proc. Natl. Acad. Sci.U.S.A. 80:21-25 (1983)), see also “Useful proteins from recombinantbacteria” in Scientific American, 242:74-94 (1980); promoter elementsfrom yeast or other fungi such as the Gal 4 promoter, the ADH (alcoholdehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkalinephosphatase promoter, and the following animal transcriptional controlregions, which exhibit tissue specificity and have been utilized intransgenic animals; elastase I gene control region which is active inpancreatic acinar cells (Swift et al., Cell 38:639-646 (1984); Ornitz etal., Cold Spring Harbor Symp. Quant. Biol. 50:399-409 (1986); MacDonald,Hepatology 7:425-515 (1987); insulin gene control region which is activein pancreatic beta cells [Hanahan, Nature 315:115-122 (1985)];immunoglobulin gene control region which is active in lymphoid cells(Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444),mouse mammary tumor virus control region which is active in testicular,breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495),albumin gene control region which is active in liver (Pinkert et al.,1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene control regionwhich is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol.5:1639-1648; Hammer et al., 1987, Science 235:53-58); alpha1-antitrypsin gene control region which is active in the liver (Kelseyet al, 1987, Genes and Devel. 1:161-171), beta-globin gene controlregion which is active in myeloid cells (Mogram et al., 1985, Nature315:338-340; Kollias et al., 1986, Cell 46:89-94); myelin basic proteingene control region which is active in oligodendrocytes in the brain(Readhead et al., 1987, Cell 48:703-712); myosin light chain-2 genecontrol region which is active in skeletal muscle (Shani, 1985, Nature314:283-286), and gonadotropic releasing hormone gene control regionwhich is active in the hypothalamus (Mason et al., 1986, Science234:1372-1378). The invention further encompasses the production ofantisense compounds which are capable of specifically hybridizing with asequence of RNA encoding a modified TIE-2 ligand to modulate itsexpression. Ecker, U.S. Pat. No. 5,166,195, issued Nov. 24, 1992.

Thus, according to the invention, expression vectors capable of beingreplicated in a bacterial or eukaryotic host comprising a nucleic acidencoding a modified TIE-2 ligand as described herein, are used totransfect a host and thereby direct expression of such nucleic acid toproduce a modified TIE-2 ligand, which may then be recovered in abiologically active form. As used herein, a biologically active formincludes a form capable of binding to TIE receptor and causing abiological response such as a differentiated function or influencing thephenotype of the cell expressing the receptor. Such biologically activeforms could, for example, induce phosphorylation of the tyrosine kinasedomain of TIE receptor. Alternatively, the biological activity may be aneffect as an antagonist to the TIE receptor. In alternative embodiments,the active form of a modified TIE-2 ligand is one that can recognize TIEreceptor and thereby act as a targeting agent for the receptor for usein both diagnostics and therapeutics. In accordance with suchembodiments, the active form need not confer upon any TIE expressingcell any change in phenotype.

Expression vectors containing the gene inserts can be identified by fourgeneral approaches: (a) DNA-DNA hybridization, (b) presence or absenceof “marker” gene functions, (c) expression of inserted sequences and (d)PCR detection. In the first approach, the presence of a foreign geneinserted in an expression vector can be detected by DNA-DNAhybridization using probes comprising sequences that are homologous toan inserted modified TIE-2 ligand encoding gene. In the second approach,the recombinant vector/host system can be identified and selected basedupon the presence or absence of certain “marker” gene functions (e.g.,thymidine kinase activity, resistance to antibiotics, transformationphenotype, occlusion body formation in baculovirus, etc.) caused by theinsertion of foreign genes in the vector. For example, if a nucleic acidencoding a modified TIE-2 ligand is inserted within the marker genesequence of the vector, recombinants containing the insert can beidentified by the absence of the marker gene function. In the thirdapproach, recombinant expression vectors can be identified by assayingthe foreign gene product expressed by the recombinant. Such assays canbe based, for example, on the physical or functional properties of amodified TIE-2 ligand gene product, for example, by binding of theligand to TIE receptor or a portion thereof which may be tagged with,for example, a detectable antibody or portion thereof or by binding toantibodies produced against the modified TIE-2 ligand protein or aportion thereof. Cells of the present invention may transiently or,preferably, constitutively and permanently express a modified TIE-2ligand as described herein. In the fourth approach, DNA nucleotideprimers can be prepared corresponding to a tie specific DNA sequence.These primers could then be used to PCR a tie gene fragment. (PCRProtocols: A Guide To Methods and Applications, Edited by Michael A.Innis et al., Academic Press (1990)).

The recombinant ligand may be purified by any technique which allows forthe subsequent formation of a stable, biologically active protein.Preferably, the ligand is secreted into the culture medium from which itis recovered. Alternatively, the ligand may be recovered from cellseither as soluble proteins or as inclusion bodies, from which it may beextracted quantitatively by 8M guanidinium hydrochloride and dialysis inaccordance with well known methodology. In order to further purify theligand, affinity chromatography, conventional ion exchangechromatography, hydrophobic interaction chromatography, reverse phasechromatography or gel filtration may be used.

In additional embodiments of the invention, as described in greaterdetail in the Examples, a modified TIE-2 ligand encoding gene may beused to inactivate or “knock out” an endogenous gene by homologousrecombination, and thereby create a TIE ligand deficient cell, tissue,or animal. For example, and not by way of limitation, the recombinantTIE ligand-4 encoding gene may be engineered to contain an insertionalmutation, for example the neo gene, which would inactivate the nativeTIE ligand-4 encoding gene. Such a construct, under the control of asuitable promoter, may be introduced into a cell, such as an embryonicstem cell, by a technique such as transfection, transduction, orinjection. Cells containing the construct may then be selected by G418resistance. Cells which lack an intact TIE ligand-4 encoding gene maythen be identified, e.g. by Southern blotting, PCR detection, Northernblotting or assay of expression. Cells lacking an intact TIE ligand-4encoding gene may then be fused to early embryo cells to generatetransgenic animals deficient in such ligand. Such an animal may be usedto define specific in vivo processes, normally dependent upon theligand.

The present invention also provides for antibodies to a modified TIE-2ligand described herein which are useful for detection of the ligand in,for example, diagnostic applications. For preparation of monoclonalantibodies directed toward a modified TIE-2 ligand, any technique whichprovides for the production of antibody molecules by continuous celllines in culture may be used. For example, the hybridoma techniqueoriginally developed by Kohler and Milstein (1975, Nature 256:495-497),as well as the trioma technique, the human B-cell hybridoma technique(Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridomatechnique to produce human monoclonal antibodies (Cole et al., 1985, in“Monoclonal Antibodies and Cancer Therapy,” Alan R. Liss, Inc. pp.77-96) and the like are within the scope of the present invention.

The monoclonal antibodies may be human monoclonal antibodies or chimerichuman-mouse (or other species) monoclonal antibodies. Human monoclonalantibodies may be made by any of numerous techniques known in the art(e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:7308-7312;Kozbor et al., 1983, Immunology Today 4:72-79; Olsson et al., 1982,Meth. Enzymol. 92:3-16). Chimeric antibody molecules may be preparedcontaining a mouse antigen-binding domain with human constant regions(Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:6851, Takeda etal., 1985, Nature 314:452).

Various procedures known in the art may be used for the production ofpolyclonal antibodies to epitopes of a modified TIE-2 ligand describedherein. For the production of antibody, various host animals, includingbut not limited to rabbits, mice and rats can be immunized by injectionwith a modified TIE-2 ligand, or a fragment or derivative thereof.Various adjuvants may be used to increase the immunological response,depending on the host species, and including but not limited to Freund's(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,dinitrophenol, and potentially useful human adjuvants such as BCG(Bacille Calmette-Guerin) and Corynebacterium parvum.

A molecular clone of an antibody to a selected a modified TIE-2 ligandepitope can be prepared by known techniques. Recombinant DNA methodology(see e.g., Maniatis et al., 1982, Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) may beused to construct nucleic acid sequences which encode a monoclonalantibody molecule, or antigen binding region thereof.

The present invention provides for antibody molecules as well asfragments of such antibody molecules. Antibody fragments which containthe idiotype of the molecule can be generated by known techniques. Forexample, such fragments include but are not limited to: the F(ab′)₂fragment which can be produced by pepsin digestion of the antibodymolecule; the Fab′ fragments which can be generated by reducing thedisulfide bridges of the F(ab′)₂ fragment, and the Fab fragments whichcan be generated by treating the antibody molecule with papain and areducing agent. Antibody molecules may be purified by known techniques,es, immunoabsorption or immunoaffinity chromatography, chromatographicmethods such as HPLC (high performance liquid chromatography), or acombination thereof.

The present invention further encompasses an immunoassay for measuringthe amount of a modified TIE-2 ligand in a biological sample by

a) contacting the biological sample with at least one antibody whichspecifically binds a modified TIE-2 ligand so that the antibody forms acomplex with any modified TIE-2 ligand present in the sample; and

b) measuring the amount of the complex and thereby measuring the amountof the modified TIE-2 ligand in the biological sample.

The invention further encompasses an assay for measuring the amount ofTIE receptor in a biological sample by

a) contacting the biological sample with at least one ligand of theinvention so that the ligand forms a complex with the TIE receptor; and

b) measuring the amount of the complex and thereby measuring the amountof the TIE receptor in the biological sample.

The present invention also provides for the utilization of a modifiedTIE-2 ligand which activates the TIE-2 receptor as described herein, tosupport the survival and/or growth and/or migration and/ordifferentiation of TIE-2 receptor expressing cells. Thus, the ligand maybe used as a supplement to support, for example, endothelial cells inculture.

Further, the creation by applicants of a modified TIE-2 ligand for theTIE-2 receptor enables the utilization of assay systems useful for theidentification of agonists or antagonists of the TIE-2 receptor. Suchassay systems would be useful in identifying molecules capable ofpromoting or inhibiting angiogenesis. For example, in one embodiment,antagonists of the TIE-2 receptor may be identified as test moleculesthat are capable of interfering with the interaction of the TIE-2receptor with a modified TIE-2 ligand that binds the TIE-2 receptor.Such antagonists are identified by their ability to 1) block the bindingof a biologically active modified TIE-2 ligand to the receptor asmeasured, for example, using BIAcore biosensor technology (BIAcore;Pharmacia Biosensor, Piscataway, N.J.); or 2) block the ability of abiologically active modified TIE-2 ligand to cause a biologicalresponse. Such biological responses include, but are not limited to,phosphorylation of the TIE receptor or downstream components of the TIEsignal transduction pathway, or survival, growth or differentiation ofTIE receptor bearing cells.

In one embodiment, cells engineered to express the TIE receptor may bedependent for growth on the addition of a modified TIE-2 ligand. Suchcells provide useful assay systems for identifying additional agonistsof the TIE receptor, or antagonists capable of interfering with theactivity of the modified TIE-2 ligand on such cells. Alternatively,autocrine cells, engineered to be capable of co-expressing both amodified TIE-2 ligand and receptor, may provide useful systems forassaying potential agonists or antagonists.

Therefore, the present invention provides for introduction of a TIE-2receptor into cells that do not normally express this receptor, thusallowing these cells to exhibit profound and easily distinguishableresponses to a ligand which binds this receptor. The type of responseelicited depends on the cell utilized, and not the specific receptorintroduced into the cell. Appropriate cell lines can be chosen to yielda response of the greatest utility for assaying, as well as discovering,molecules that can act on tyrosine kinase receptors. The molecules maybe any type of molecule, including but not limited to peptide andnon-peptide molecules, that will act in systems to be described in areceptor specific manner.

One of the more useful systems to be exploited involves the introductionof a TIE receptor (or a chimeric receptor comprising the extracellulardomain of another receptor tyrosine kinase such as, for example, trkCand the intracellular domain of a TIE receptor) into a fibroblast cellline (e.g., NIH3T3 cells) thus such a receptor which does not normallymediate proliferative or other responses can, following introductioninto fibroblasts, nonetheless be assayed by a variety of wellestablished methods to quantitate effects of fibroblast growth factors(e.g. thymidine incorporation or other types of proliferation assays;see van Zoelen, 1990, “The Use of Biological Assays For Detection OfPolypeptide Growth Factors” in Progress Factor Research, Vol. 2, pp.131-152; Zhan and M. Goldfarb, 1986, Mol. Cell. Biol., Vol. 6, pp.3541-3544). These assays have the added advantage that any preparationcan be assayed both on the cell line having the introduced receptor aswell as the parental cell line lacking the receptor; only specificeffects on the cell line with the receptor would be judged as beingmediated through the introduced receptor. Such cells may be furtherengineered to express a modified TIE-2 ligand, thus creating anautocrine system useful for assaying for molecules that act asantagonists/agonists of this interaction. Thus, the present inventionprovides for host cells comprising nucleic acid encoding a modifiedTIE-2 ligand and nucleic acid encoding TIE receptor.

The TIE receptor/modified TIE-2 ligand interaction also provides auseful system for identifying small molecule agonists or antagonists ofthe TIE receptor. For example, fragments, mutants or derivatives of amodified TIE-2 ligand may be identified that bind TIE receptor but donot induce any other biological activity. Alternatively, thecharacterization of a modified TIE-2 ligand enables the furthercharacterization of active portions of the molecule. Further, theidentification of a ligand enables the determination of the X-raycrystal structure of the receptor/ligand complex, thus enablingidentification of the binding site on the receptor. Knowledge of thebinding site will provide useful insight into the rational design ofnovel agonists and antagonists.

The specific binding of a test molecule to TIE receptor may be measuredin a number of ways. For example, the actual binding of test molecule tocells expressing TIE may be detected or measured, by detecting ormeasuring (i) test molecule bound to the surface of intact cells; (ii)test molecule cross-linked to TIE protein in cell lysates; or (iii) testmolecule bound to TIE in vitro. The specific interaction between testmolecule and TIE may be evaluated by using reagents that demonstrate theunique properties of that interaction.

As a specific, nonlimiting example, the methods of the invention may beused as follows. Consider a case in which a modified TIE-2 ligand in asample is to be measured. Varying dilutions of the sample (the testmolecule), in parallel with a negative control (NC) containing nomodified TIE-2 ligand activity, and a positive control (PC) containing aknown amount of a modified TIE-2 ligand, may be exposed to cells thatexpress TIE in the presence of a detectably labeled modified TIE-2ligand (in this example, radioiodinated ligand). The amount of modifiedTIE-2 ligand in the test sample may be evaluated by determining theamount of ¹²⁵I-labeled modified TIE-2 ligand that binds to the controlsand in each of the dilutions, and then comparing the sample values to astandard curve. The more modified TIE-2 ligand in the sample, the less¹²⁵I-ligand that will bind to TIE.

The amount of ¹²⁵I-ligand bound may be determined by measuring theamount of radioactivity per cell, or by cross-linking a modified TIE-2ligand to cell surface proteins using DSS, as described in Meakin andShooter, 1991, Neuron 6:153-163, and detecting the amount of labeledprotein in cell extracts using, for example, SDS polyacrylamide gelelectrophoresis, which may reveal a labeled protein having a sizecorresponding to TIE receptor/modified TIE-2 ligand. The specific testmolecule/TIE interaction may further be tested by adding to the assaysvarious dilutions of an unlabeled control ligand that does not bind theTIE receptor and therefore should have no substantial effect on thecompetition between labeled modified TIE-2 ligand and test molecule forTIE binding. Alternatively, a molecule known to be able to disrupt TIEreceptor/modified TIE-2 ligand binding, such as, but not limited to,anti-TIE antibody, or TIE receptorbody as described herein, may beexpected to interfere with the competition between ¹²⁵I-modified TIE-2ligand and test molecule for TIE receptor binding.

Detectably labeled modified TIE-2 ligand includes, but is not limitedto, a modified TIE-2 ligand linked covalently or noncovalently to aradioactive substance, a fluorescent substance, a substance that hasenzymatic activity, a substance that may serve as a substrate for anenzyme (enzymes and substrates associated with colorimetricallydetectable reactions are preferred) or to a substance that can berecognized by an antibody molecule that is preferably a detectablylabeled antibody molecule.

Alternatively, the specific binding of test molecule to TIE may bemeasured by evaluating the secondary biological effects of a modifiedTIE-2 ligand/TIE receptor binding, including, but not limited to, cellgrowth and/or differentiation or immediate early gene expression orphosphorylation of TIE. For example, the ability of the test molecule toinduce differentiation can be tested in cells that lack tie and incomparable cells that express tie; differentiation in tie-expressingcells but not in comparable cells that lack tie would be indicative of aspecific test molecule/TIE interaction. A similar analysis could beperformed by detecting immediate early gene (e.g. fos and jun) inductionin tie-minus and tie-plus cells, or by detecting phosphorylation of TIEusing standard phosphorylation assays known in the art. Such analysismight be useful in identifying agonists or antagonists that do notcompetitively bind to TIE.

Similarly, the present invention provides for a method of identifying amolecule that has the biological activity of a modified TIE-2 ligandcomprising (i) exposing a cell that expresses tie to a test molecule and(ii) detecting the specific binding of the test molecule to TIEreceptor, in which specific binding to TIE positively correlates withTIE-like activity. Specific binding may be detected by either assayingfor direct binding or the secondary biological effects of binding, asdiscussed supra. Such a method may be particularly useful in identifyingnew members of the TIE ligand family or, in the pharmaceutical industry,in screening a large array of peptide and non-peptide molecules (e.g.,peptidomimetics) for TIE associated biological activity. In a preferred,specific, nonlimiting embodiment of the invention, a large grid ofculture wells may be prepared that contain, in alternate rows, PC12 (orfibroblasts, see infra) cells that are either tie-minus or engineered tobe tie-plus. A variety of test molecules may then be added such thateach column of the grid, or a portion thereof, contains a different testmolecule. Each well could then be scored for the presence or absence ofgrowth and/or differentiation. An extremely large number of testmolecules could be screened for such activity in this manner.

In additional embodiments, the invention provides for methods ofdetecting or measuring TIE ligand-like activity or identifying amolecule as having such activity comprising (i) exposing a test moleculeto a TIE receptor protein in vitro under conditions that permit bindingto occur and (ii) detecting binding of the test molecule to the TIEreceptor protein, in which binding of test molecule to TIE receptorcorrelates with TIE ligand-like activity. According to such methods, theTIE receptor may or may not be substantially purified, may be affixed toa solid support (e.g. as an affinity column or as an ELISA assay), ormay be incorporated into an artificial membrane. Binding of testmolecule to TIE receptor may be evaluated by any method known in theart. In preferred embodiments, the binding of test molecule may bedetected or measured by evaluating its ability to compete withdetectably labeled known TIE ligands for TIE receptor binding.

The present invention also provides for a method of detecting theability of a test molecule to function as an antagonist of TIEligand-like activity comprising detecting the ability of the molecule toinhibit an effect of TIE ligand binding to TIE receptor on a cell thatexpresses the receptor. Such an antagonist may or may not interfere withTIE receptor/modified TIE-2 ligand binding. Effects of a modified TIE-2ligand binding to TIE receptor are preferably biological or biochemicaleffects, including, but not limited to, cell survival or proliferation,cell transformation, immediate early gene induction, or TIEphosphorylation.

The invention further provides for both a method of identifyingantibodies or other molecules capable of neutralizing the ligand orblocking binding to the receptor, as well as the molecules identified bythe method. By way of nonlimiting example, the method may be performedvia an assay which is conceptually similar to an ELISA assay. Forexample, TIE receptorbody may be bound to a solid support, such as aplastic multiwell plate. As a control, a known amount of a modifiedTIE-2 ligand which has been Myc-tagged may then be introduced to thewell and any tagged modified TIE-2 ligand which binds the receptorbodymay then be identified by means of a reporter antibody directed againstthe Myc-tag. This assay system may then be used to screen test samplesfor molecules which are capable of i) binding to the tagged ligand orii) binding to the receptorbody and thereby blocking binding to thereceptorbody by the tagged ligand. For example, a test sample containinga putative molecule of interest together with a known amount of taggedligand may be introduced to the well and the amount of tagged ligandwhich binds to the receptorbody may be measured. By comparing the amountof bound tagged ligand in the test sample to the amount in the control,samples containing molecules which are capable of blocking ligandbinding to the receptor may be identified. The molecules of interestthus identified may be isolated using methods well known to one of skillin the art.

Once a blocker of ligand binding is found, one of skill in the art wouldknow to perform secondary assays to determine whether the blocker isbinding to the receptor or to the ligand, as well as assays to determineif the blocker molecule can neutralize the biological activity of theligand. For example, by using a binding assay which employs BIAcorebiosensor technology (or the equivalent), in which either TIEreceptorbody or a modified TIE-2 ligand or ligandbody is covalentlyattached to a solid support (e.g. carboxymethyl dextran on a goldsurface), one of skill in the art would be able to determine if theblocker molecule is binding specifically to the ligand, ligandbody or tothe receptorbody. To determine if the blocker molecule can neutralizethe biological activity of the ligand, one of skill in the art couldperform a phosphorylation assay (see Example 5) or alternatively, afunctional bioassay, such as a survival assay, by using primary culturesof, for example, endothelial cells. Alternatively, a blocker moleculewhich binds to the receptorbody could be an agonist and one of skill inthe art would know to how to determine this by performing an appropriateassay for identifying additional agonists of the TIE receptor.

In addition, the invention further contemplates compositions wherein theTIE ligand is the receptor binding domain of a TIE-2 ligand describedherein. For example, TIE-2 ligand 1 contains a “coiled coil” domain(beginning at the 5′ end and extending to the nucleotide at aboutposition 1160 of FIGS. 4A-4D [SEQ ID NO: 1 and SEQ ID NO: 2] and aboutposition 1157 of FIGS. 5A-5D [SEQ ID NO: 3 and SEQ ID NO: 4]) and afibrinogen-like domain (which is encoded by the nucleotide sequence ofFIGS. 4A-4D [SEQ ID NO: 1 and SEQ ID NO: 2] beginning at about position1161 and about position 1158 of FIGS. 5A-5D [SEQ ID NO: 3 and SEQ ID NO:4]). The fibrinogen-like domain of TIE-2 ligand 2 is believed to beginon or around the same amino acid sequence as in ligand 1 (FRDCA) whichis encoded by nucleotides beginning around 1197 of FIGS. 6A-6D (SEQ IDNO: 5 and SEQ ID NO: 6). The fibrinogen-like domain of TIE ligand-3 isbelieved to begin on or around the amino acid sequence which is encodedby nucleotides beginning around position 929 as set forth in FIGS.21A-21C (SEQ ID NO: 9 and SEQ ID NO: 10). Multimerization of the coiledcoil domains during production of the ligand hampers purification. Asdescribed in Example 19, Applicants have discovered, however, that thefibrinogen-like domain comprises the TIE-2 receptor binding domain. Themonomeric forms of the fibrinogen-like domain do not, however, appear tobind the receptor. Studies utilizing myc-tagged fibrinogen-like domain,which has been “clustered” using anti-myc antibodies, do bind the TIE-2receptor. [Methods of production of “clustered ligands and ligandbodiesare described in Davis, et al. Science 266:816-819 (1994)]. Based onthese finding, applicants produced “ligandbodies” which comprise thefibrinogen-like domain of the TIE-2 ligands coupled to the Fc domain ofIgG (“fFc's”). These ligandbodies, which form dimers, efficiently bindthe TIE-2 receptor. Accordingly, the present invention contemplates theproduction of modified TIE ligandbodies which may be used as targetingagents, in diagnostics or in therapeutic applications, such as targetingagents for tumors and/or associated vasculature wherein a TIE antagonistis indicated.

The invention herein further provides for the development of the ligand,a fragment or derivative thereof, or another molecule which is areceptor agonist or antagonist, as a therapeutic for the treatment ofpatients suffering from disorders involving cells, tissues or organswhich express the TIE receptor. Such molecules may be used in a methodof treatment of the human or animal body, or in a method of diagnosis.

Because TIE receptor has been identified in association with endothelialcells and, as demonstrated herein, blocking of TIE-2 ligand 1 appears toprevent vascularization, applicants expect that a modified TIE-2 liganddescribed herein may be useful for the induction of vascularization indiseases or disorders where such vascularization is indicated. Suchdiseases or disorders would include wound healing, ischaemia anddiabetes. The ligands may be tested in animal models and usedtherapeutically as described for other agents, such as vascularendothelial growth factor (VEGF), another endothelial cell-specificfactor that is angiogenic. Ferrara, et al. U.S. Pat. No. 5,332,671issued Jul. 26, 1994. The Ferrara reference, as well as other studies,describe in vitro and in vivo studies that may be used to demonstratethe effect of an angiogenic factor in enhancing blood flow to ischemicmyocardium, enhancing wound healing, and in other therapeutic settingswherein neoangiogenesis is desired. [see Sudo, et al. European PatentApplication 0 550 296 A2 published Jul. 7, 1993; Banai, et al.Circulation 89:2183-2189 (1994); Unger, et al. Am. J. Physiol.266:H1588-H1595 (1994); Lazarous, et al. Circulation 91:145-153 (1995)].According to the invention, a modified TIE-2 ligand may be used alone orin combination with one or more additional pharmaceutically activecompounds such as, for example, VEGF or basic fibroblast growth factor(bFGF), as well as cytokines, neurotrophins, etc.

Conversely, antagonists of the TIE receptor, such as modified TIE-2ligands which bind but do not activate the receptor as described herein,receptorbodies as described herein in Examples 2 and 3, and TIE-2 ligand2 as described in Example 9, would be useful to prevent or attenuatevascularization, thus preventing or attenuating, for example, tumorgrowth. These agents may be used alone or in combination with othercompositions, such as anti-VEGF antibodies, that have been shown to beuseful in treating conditions in which the therapeutic intent is toblock angiogenesis. Applicants expect that a modified TIE-2 liganddescribed herein may also be used in combination with agents, such ascytokine antagonists such as IL-6 antagonists, that are known to blockinflammation.

For example, applicants have determined that TIE ligands are expressedin cells within, or closely associated with, tumors. For example, TIE-2ligand 2 appears to be tightly associated with tumor endothelial cells.Accordingly, it and other TIE antagonists may also be useful inpreventing or attenuating, for example, tumor growth. In addition, TIEligands or ligandbodies may be useful for the delivery of toxins to areceptor bearing cell. Alternatively, other molecules, such as growthfactors, cytokines or nutrients, may be delivered to a TIE receptorbearing cell via TIE ligands or ligandbodies. TIE ligands orligandbodies such as modified TIE-2 ligand described herein may also beused as diagnostic reagents for TIE receptor, to detect the receptor invivo or in vitro. Where the TIE receptor is associated with a diseasestate, TIE ligands or ligandbodies such as a modified TIE-2 ligand maybe useful as diagnostic reagents for detecting the disease by, forexample, tissue staining or whole body imaging. Such reagents includeradioisotopes, flurochromes, dyes, enzymes and biotin. Such diagnosticsor targeting agents may be prepared as described in Alitalo, et al. WO95/26364 published Oct. 5, 1995 and Burrows, F. and P. Thorpe, PNAS(USA) 90:8996-9000 (1993) which is incorporated herein in its entirety.

In other embodiments, the TIE ligands, a receptor activating modifiedTIE-2 ligand described herein are used as hematopoietic factors. Avariety of hematopoietic factors and their receptors are involved in theproliferation and/or differentiation and/or migration of the variouscells types contained within blood. Because the TIE receptors areexpressed in early hematopoietic cells, the TIE ligands are expected toplay a comparable role in the proliferation or differentiation ormigration of these cells. Thus, for example, TIE containing compositionsmay be prepared, assayed, examined in in vitro and in vivo biologicalsystems and used therapeutically as described in any of the following:Sousa, U.S. Pat. No. 4,810,643, Lee, et al., Proc. Natl. Acad. Sci. USA82:4360-4364 (1985) Wong, et al. Science, 228:810-814 (1985); Yokota, etal. Proc. Natl. Acad. Sci (USA) 81:1070 (1984); Bosselman, et al. WO9105795 published May 2, 1991 entitled “Stem Cell Factor” and Kirkness,et al. WO 95/19985 published Jul. 27, 1995 entitled “HaemopoieticMaturation Factor”. Accordingly, receptor activating modified TIE-2ligand may be used to diagnose or treat conditions in which normalhematopoiesis is suppressed, including, but not limited to anemia,thrombocytopenia, leukopenia and granulocytopenia. In a preferredembodiment, receptor activating modified TIE-2 ligand may be used tostimulate differentiation of blood cell precursors in situations where apatient has a disease, such as acquired immune deficiency syndrome(AIDS) which has caused a reduction in normal blood cell levels, or inclinical settings in which enhancement of hematopoietic populations isdesired, such as in conjunction with bone marrow transplant, or in thetreatment of aplasia or myelosuppression caused by radiation, chemicaltreatment or chemotherapy.

The receptor activating modified TIE-2 ligands of the present inventionmay be used alone, or in combination with another pharmaceuticallyactive agent such as, for example, ctyokines, neurotrophins,interleukins, etc. In a preferred embodiment, the ligands may be used inconjunction with any of a number of the above referenced factors whichare known to induce stem cell or other hematopoietic precursorproliferation, or factors acting on later cells in the hematopoieticpathway, including, but not limited to, hemopoietic maturation factor,thrombopoietin, stem cell factor, erythropoietin, G-CSF, GM-CSF, etc.

In an alternative embodiment, TIE receptor antagonists are used todiagnose or treat patients in which the desired result is inhibition ofa hematopoietic pathway, such as for the treatment of myeloproliferativeor other proliferative disorders of blood forming organs such asthrombocythemias, polycythemias and leukemias. In such embodiments,treatment may comprise use of a therapeutically effective amount of thea modified TIE-2 ligand, TIE antibody, TIE receptorbody, a conjugate ofa modified TIE-2 ligand, or a ligandbody or fFC as described herein.

The present invention also provides for pharmaceutical compositionscomprising a modified TIE-2 ligand or ligandbodies described herein,peptide fragments thereof, or derivatives in a pharmacologicallyacceptable vehicle. The modified TIE-2 ligand proteins, peptidefragments, or derivatives may be administered systemically or locally.Any appropriate mode of administration known in the art may be used,including, but not limited to, intravenous, intrathecal, intraarterial,intranasal, oral, subcutaneous, intraperitoneal, or by local injectionor surgical implant. Sustained release formulations are also providedfor.

The present invention also provides for an antibody which specificallybinds such a therapeutic molecule. The antibody may be monoclonal orpolyclonal. The invention also provides for a method of using such amonoclonal or polyclonal antibody to measure the amount of thetherapeutic molecule in a sample taken from a patient for purposes ofmonitoring the course of therapy.

The invention further provides for a therapeutic composition comprisinga modified TIE-2 ligand or ligandbody and a cytotoxic agent conjugatedthereto. In one embodiment, the cytotoxic agent may be a radioisotope ortoxin.

The invention also provides for an antibody which specifically binds amodified TIE-2 ligand. The antibody may be monoclonal or polyclonal. Theinvention further provides for a method of purifying a modified TIE-2ligand comprising:

a) coupling at least one TIE binding substrate to a solid matrix;

b) incubating the substrate of a) with a cell lysate so that thesubstrate forms a complex with any modified TIE-2 ligand in the celllysate;

c) washing the solid matrix; and

d) eluting the modified TIE-2 ligand from the coupled substrate.

The substrate may be any substance that specifically binds the modifiedTIE-2 ligand. In one embodiment, the substrate is selected from thegroup consisting of anti-modified TIE-2 ligand antibody, TIE receptorand TIE receptorbody. The invention further provides for a receptorbodywhich specifically binds a modified TIE-2 ligand, as well as atherapeutic composition comprising the receptorbody in apharmaceutically acceptable vehicle, and a method of blocking bloodvessel growth in a human comprising administering an effective amount ofthe therapeutic composition.

The invention also provides for a therapeutic composition comprising areceptor activating modified TIE-2 ligand or ligandbody in apharmaceutically acceptable vehicle, as well as a method of promotingneovascularization in a patient comprising administering to the patientan effective amount of the therapeutic composition.

In addition, the present invention provides for a method for identifyinga cell which expresses TIE receptor which comprises contacting a cellwith a detectably labeled modified TIE-2 ligand or ligandbody, underconditions permitting binding of the detectably labeled ligand to theTIE receptor and determining whether the detectably labeled ligand isbound to the TIE receptor, thereby identifying the cell as one whichexpresses TIE receptor. The present invention also provides for atherapeutic composition comprising a modified TIE-2 ligand or ligandbodyand a cytotoxic agent conjugated thereto. The cytotoxic agent may be aradioisotope or toxin.

The invention also provides a method of detecting expression of amodified TIE-2 ligand by a cell which comprises obtaining mRNA from thecell, contacting the mRNA so obtained with a labeled nucleic acidmolecule encoding a modified TIE-2 ligand, under hybridizing conditions,determining the presence of mRNA hybridized to the labeled molecule, andthereby detecting the expression of a modified TIE-2 ligand in the cell.

The invention further provides a method of detecting expression of amodified TIE-2 ligand in tissue sections which comprises contacting thetissue sections with a labeled nucleic acid molecule encoding a modifiedTIE-2 ligand, under hybridizing conditions, determining the presence ofmRNA hybridized to the labelled molecule, and thereby detecting theexpression of a modified TIE-2 ligand in tissue sections.

EXAMPLE 1 Identification of the ABAE Cell Line as Reporter Cells for theTIE-2 Receptor

Adult BAE cells are registered in the European Cell Culture Repository,under ECACC#92010601. (See PNAS 75:2621 (1978)). Northern (RNA) analysesrevealed moderate levels of tie-2 transcripts in the ABAE (Adult BovineArterial Endothelial) cell line, consistent with in situ hybridizationresults that demonstrated almost exclusive localization of tie-2 RNAs tovascular endothelial cells. We therefore examined ABAE cell lysates forthe presence of TIE-2 protein, as well as the extent to which this TIE-2protein is tyrosine-phosphorylated under normal versus serum-deprivedgrowth conditions. ABAE cell lysates were harvested and subjected toimmunoprecipitation, followed by Western blot analyses ofimmunoprecipitated proteins with TIE-2 specific andphosphotyrosine-specific antisera. Omission or inclusion of TIE-2peptides as specific blocking molecules during TIE-2 immunoprecipitationallowed unambiguous identification of TIE-2 as a moderately detectableprotein of ˜150 kD whose steady-state phosphotyrosine levels diminish tonear undetectable levels by prior serum-starvation of the cells.

Culture of ABAE cells and harvest of cell lysates was done as follows.Low-passage-number ABAE cells were plated as a monolayer at a density of2×10⁶ cells/150 mm plastic petri plate (Falcon) and cultured inDulbecco's modified Eagle's medium (DMEM) containing 10% bovine calfserum (10% BCS), 2 mM L-glutamine (Q) and 1% each of penicillin andstreptomycin (P-S) in an atmosphere of 5% CO₂. Prior to harvest of celllysates, cells were serum-starved for 24 hours in DMEM/Q/P-S, followedby aspiration of the medium and rinsing of the plates with ice-coldphosphate buffered saline (PBS) supplemented with sodium orthovanadate,sodium fluoride and sodium benzamidine. Cells were lysed in a smallvolume of this rinse buffer that had been supplemented with 1% NP40detergent and the protease inhibitors PMSF and aprotinin. Insolubledebris was removed from the cell Iysates by centrifugation at 14,000×Gfor 10 minutes, at 4° C. and the supernatants were subjected toimmunoprecipitation with antisera specific for TIE-2 receptor, with orwithout the presence of blocking peptides added to ˜20 μg/ml lysate.Immunoprecipitated proteins were resolved by PAGE (7.5% Laemmli gel),and then electro-transferred to PVDF membrane and incubated either withvarious TIE-2- or phosphotyrosine-specific antisera. TIE-2 protein wasvisualized by incubation of the membrane with HRP-linked secondaryantisera followed by treatment with ECL reagent (Amersham).

EXAMPLE 2 Cloning and Expression of TIE-2 Receptorbody forAffinity-based Study of TIE-2 Ligand Interactions

An expression construct was created that would yield a secreted proteinconsisting of the entire extracellular portion of the rat TIE-2 receptorfused to the human immunoglobulin gamma-1 constant region (IgG1 Fc).This fusion protein is called a TIE-2 “receptorbody” (RB), and would benormally expected to exist as a dimer in solution based on formation ofdisulfide linkages between individual IgG1 Fc tails. The Fc portion ofthe TIE-2 RB was prepared as follows. A DNA fragment encoding the Fcportion of human IgG1 that spans from the hinge region to thecarboxy-terminus of the protein, was amplified from human placental cDNAby PCR with oligonucleotides corresponding to the published sequence ofhuman IgG1; the resulting DNA fragment was cloned in a plasmid vector.Appropriate DNA restriction fragments from a plasmid encoding thefull-length TIE-2 receptor and from the human IgG1 Fc plasmid wereligated on either side of a short PCR-derived fragment that was designedso as to fuse, in-frame, the TIE-2 and human IgG1 Fc protein-codingsequences. Thus, the resulting TIE-2 ectodomain-Fc fusion proteinprecisely substituted the IgG1 Fc in place of the region spanning theTIE-2 transmembrane and cytoplasmic domains. An alternative method ofpreparing RBs is described in Goodwin, et. al. Cell 73:447-456 (1993).

Milligram quantities of TIE-2 RB were obtained by cloning the TIE-2 RBDNA fragment into the pVL1393 baculovirus vector and subsequentlyinfecting the Spodoptera frugiperda SF-21AE insect cell line.Alternatively, the cell line SF-9 (ATCC Accession No. CRL-1711) or thecell line BTI-TN-5b1-4 may be used. DNA encoding the TIE-2 RB was clonedas an Eco RI-NotI fragment into the baculovirus transfer plasmidpVL1393. Plasmid DNA purified by cesium chloride density gradientcentrifugation was recombined into viral DNA by mixing 3 μg of plasmidDNA with 0.5 μg of Baculo-Gold DNA (Pharminigen), followed byintroduction into liposomes using 30 μg Lipofectin (GIBCO-BRL).DNA-liposome mixtures were added to SF-21AE cells (2×10⁶ cells/60 mmdish) in TMN-FH medium (Modified Grace's Insect Cell Medium (GIBCO-BRL)for 5 hours at 27° C., followed by incubation at 27° C. for 5 days inTMN-FH medium supplemented with 5% fetal calf serum. Tissue culturemedium was harvested for plaque purification of recombinant viruses,which was carried out using methods previously described (O'Reilly, D.R., L. K. Miller, and V. A. Luckow, Baculovirus Expression Vectors—ALaboratory Manual. 1992, New York: W. H. Freeman) except that theagarose overlay contained 125 μg/mL X-gal(5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside; GIBCO-BRL). After 5days of incubation at 27° C., non-recombinant plaques were scored bypositive chromogenic reaction to the X-gal substrate, and theirpositions marked. Recombinant plaques were then visualized by additionof a second overlay containing 100 μg/mL MTT(3-[4,5-dimethylthiazol-2-yl]2,5,diphenyltetrazolium bromide; Sigma).Putative recombinant virus plaques were picked by plug aspiration, andpurified by multiple rounds of plaque isolation to assure homogeneity.Virus stocks were generated by serial, low-multiplicity passage ofplaque-purified virus. Low passage stocks of one virus clone (vTIE-2receptorbody) were produced.

SF-21AE cells were cultured in serum free medium (SF-900 II, Gibco BRL)containing 1× antibiotic/antimycotic solution (Gibco BRL) and 25 mg/LGentamycin (Gibco BRL). Pluronic F-68 was added as a surfactant to afinal concentration of 1 g/L. Cultures (4L) were raised in a bioreactor(Artisan Cell Station System) for at least three days prior toinfection. Cells were grown at 27° C., with gassing to 50% dissolvedoxygen, at a gas flow rate of 80 mL/min (aeration at a sparge ring).Agitation was by means of a marine impeller at a rate of 100 rpm. Cellswere harvested in mid-logarithmic growth phase (˜2×10⁶ cells/mL),concentrated by centrifugation, and infected with 5 plaque forming unitsof vTIE-2 receptorbody per cell. Cells and inoculum were brought to 400mL with fresh medium, and virus was adsorbed for 2 hours at 27° C. in aspinner flask. The culture was then resuspended in a final volume of 8Lwith fresh serum-free medium, and the cells incubated in the bioreactorusing the previously described conditions.

Culture medium from vTIE-2 receptorbody-infected SF21AE cells werecollected by centrifugation (500×g, 10 minutes) at 72 hourspost-infection. Cell supernatants were brought to pH 8 with NaOH. EDTAwas added to a final concentration of 10 mM and the supernatant pH wasreadjusted to 8. Supernatants were filtered (0.45 μm, Millipore) andloaded on a protein A column (protein A sepharose 4 fast flow or HiTrapprotein A, both from Pharmacia). The column was washed with PBScontaining 0.5 M NaCl until the absorbance at 280 nm decreased tobaseline. The column was washed in PBS and eluted with 0.5 M aceticacid. Column fractions were immediately neutralized by eluting intotubes containing 1 M Tris pH 9. The peak fractions containing the TIE-2receptorbody were pooled and dialyzed versus PBS.

EXAMPLE 3 Demonstration that TIE-2 has a Critical Role in Development ofthe Vasculature

Insight into the function of TIE-2 was gained by introduction of“excess” soluble TIE-2 receptorbody (TIE-2 RB) into a developing system.The potential ability of TIE-2 RB to bind, and thereby neutralize,available TIE-2 ligand could result in an observable disruption ofnormal vascular development and characterization of the ligand. Toexamine whether TIE-2 RB could be used to disrupt vascular developmentin early chick embryos, small pieces of a biologically resorbable foamwere soaked with TIE-2 RB and inserted immediately beneath thechorioallantoic membrane at positions just lateral to the primitiveembryo.

Early chicken embryos develop atop the yolk from a small disk of cellsthat is covered by the chorioallantoic membrane (CAM). The endothelialcells that will come to line the vasculature in the embryo arise fromboth extra- and intra-embryonic cell sources.Extra-embryonically-derived endothelial cells, which provide the majorsource of endothelial cells in the embryo, originate from accretions ofmesenchyme that are situated laterally around the embryo-proper, justunderneath the CAM. As these mesenchyme cells mature, they give rise toa common progenitor of both the endothelial and hematopoietic celllineages, termed the hemangioblast. In turn, the hemangioblast givesrise to a mixed population of angioblasts (the endothelial cellprogenitor) and hematoblasts (the pluripotential hematopoieticprecursor). Formation of rudiments of the circulatory system begins whenendothelial cell progeny segregate to form a one-cell-thick vesicle thatsurrounds the primitive blood cells. Proliferation and migration ofthese cellular components eventually produces a vast network ofblood-filled microvessels under the CAM that will ultimately invade theembryo to join with limited, intra-embryonically-derived vascularelements.

Newly fertilized chicken eggs obtained from Spafas, Inc. (Boston, Mass.)were incubated at 99.5° F., 55% relative humidity. At about 24 hrs. ofdevelopment, the egg shell was wiped down with 70% ethanol and adentist's drill was used to make a 1.5 cm. hole in the blunt apex ofeach egg. The shell membrane was removed to reveal an air space directlyabove the embryo. Small rectangular pieces of sterile Gelfoam (Upjohn)were cut with a scalpel and soaked in equal concentrations of eitherTIE-2- or EHK-1 receptorbody. EHK-1 receptorbody was made as set forthin Example 2 using the EHK-1 extracellular domain instead of the TIE-2extracellular domain (Maisonpierre et al., Oncogene 8:3277-3288 (1993).Each Gelfoam piece absorbed approximately 6 μg of protein in 30 μl.Sterile watchmakers forceps were used to make a small tear in the CAM ata position several millimeters lateral to the primitive embryo. Themajority of the piece of RB-soaked Gelfoam was inserted under the CAMand the egg shell was sealed over with a piece of adhesive tape. Othersimilarly-staged eggs were treated in parallel with RB of the unrelated,neuronally expressed receptor tyrosine kinase, EHK-1 (Maisonpierre etal., Oncogene 8:3277-3288 (1993). Development was allowed to proceed for4 days and then the embryos were examined by visual inspection. Embryoswere removed by carefully breaking the shells in dishes of warmed PBSand carefully cutting away the embryo with surrounding CAM. Of 12 eggstreated with each RB, 6 TIE-2 RB and 5 EHK-1 RB treated embryos haddeveloped beyond the stage observed at the start of the experiment. Adramatic difference was seen between these developed embryos, as shownin FIGS. 1A and 1B. Those treated with EHK-1 RB appeared to havedeveloped relatively normally. Four out of five EHK-1 embryos wereviable as judged by the presence of a beating heart. Furthermore, theextra-embryonic vasculature, which is visually obvious due to thepresence of red blood cells, was profuse and extended severalcentimeters laterally under the CAM. By contrast, those treated withTIE-2 RB were severely stunted, ranging from 2-5 mm. in diameter, ascompared with more than 10 mm in diameter for the EHK-1 RB embryos. Allof the TIE-2 RB treated embryos were dead and their CAMs were devoid ofblood vessels. The ability of TIE-2 RB to block vascular development inthe chicken demonstrates that TIE-2 ligand is necessary for developmentof the vasculature.

EXAMPLE 4 Identification of a TIE-2-Specific Binding Activity inConditioned Medium from the ras Oncogene-Transformed C2C12 MouseMyoblast Cell Line

Screening of ten-fold-concentrated cell-conditioned media (10× CCM) fromvarious cell lines for the presence of soluble, TIE-2-specific bindingactivity (BIAcore; Pharmacia Biosensor, Piscataway, N.J.) revealedbinding activity in serum-free medium from oncogenic-ras-transformedC2C12 cells (C2C12-ras), RAT 2-ras (which is a ras transformedfibroblast cell line), human glioblastoma T98G and the humanneuroblastoma cell line known as SHEP-1.

The C2C12-ras 10× CCM originated from a stably transfected line of C2C12myoblasts that was oncogenically transformed by transfection with theT-24 mutant of H-ras by standard calcium phosphate-based methods. AnSV40 based neomycin-resistance expression plasmid was physically linkedwith the ras expression plasmid in order to permit selection oftransfected clones. Resulting G418-resistant ras-C2C12 cells wereroutinely maintained as a monolayer on plastic dishes inDMEM/glutamine/penicillin-streptomycin supplemented with 10% fetal calfserum (FCS). Serum-free C2C12-ras 10× CCM was made by plating the cellsat 60% confluence in a serum free defined media for 12 hours. [Zhan andGoldfarb, Mol. Cell. Biol. 6: 3541-3544 (1986)); Zhan, et al. Oncogene1: 369-376 (1987)]. The medium was discarded and replaced with freshDMEM/Q/P-S for 24 hours. This medium was harvested and cells were re-fedfresh DMEM/Q/P-S, which was also harvested after a further 24 hours.These CCM were supplemented with the protease inhibitors PMSF (1 mM) andaprotinin (10 μg/ml), and ten-fold concentrated on sterilesize-exclusion membranes (Amicon). TIE-2-binding activity could beneutralized by incubation of the medium with an excess of TIE-2 RB, butnot by incubation with EHK-1 RB, prior to BIAcore analysis.

Binding activity of the 10× CCM was measured using biosensor technology(BIAcore; Pharmacia Biosensor, Piscataway, N.J.) which monitorsbiomolecular interactions in real-time via surface plasmon resonance.Purified TIE-2 RB was covalently coupled through primary amines to thecarboxymethyl dextran layer of a CM5 research grade sensor chip(Pharmacia Biosensor; Piscataway, N.J.). The sensor chip surface wasactivated using a mixture of N-hydroxysuccinimide (NHS) andN-ethyl-N′-(3-dimethylaminopropyl)carbodiimide (EDC), followed byimmobilization of TIE-2 RB (25 μg/mL, pH 4.5) and deactivation ofunreacted sites with 1.0 M ethanolamine (pH 8.5). A negative controlsurface of the EHK-1 receptorbody was prepared in a similar manner.

The running buffer used in the system was HBS (10 mM Hepes, 3.4 mM EDTA,150 mM NaCl, 0.005% P20 surfactant, pH 7.4). The 10× CCM samples werecentrifuged for 15 min at 4° C. and further clarified using a sterile,low protein-binding 0.45 μm filter (Millipore; Bedford, Mass.). Dextran(2 mg/ml) and P20 surfactant (0.005%) were added to each CCM sample.Aliquots of 40 μL were injected across the immobilized surface (eitherTIE-2 or EHK-1) at a flow rate of 5 μL/min and the receptor binding wasmonitored for 8 min. The binding activity (resonance units, RU) wasmeasured as the difference between a baseline value determined 30 sprior to the sample injection and a measurement taken at 30 spost-injection. Regeneration of the surface was accomplished with one12-μL pulse of 3 M MgCl₂.

The instrument noise level is 20 RU; therefore, any binding activitywith a signal above 20 RU may be interpreted as a real interaction withthe receptor. For C2C12-ras conditioned media, the binding activitieswere in the range 60-90 RU for the TIE-2 RB immobilized surface. For thesame samples assayed on a EHK-1 RB immobilized surface, the measuredactivities were less than 35 RU. Specific binding to the TIE-2receptorbody was evaluated by incubating the samples with an excess ofeither soluble TIE-2 or EHK-1 RB prior to assaying the binding activity.The addition of soluble EHK-1 RB had no effect on the TIE-2 bindingactivity of any of the samples, while in the presence of soluble TIE-2binding to the surface is two-thirds less than that measured in theabsence of TIE-2. A repeat assay using >50× concentrated C2C12-ras CCMresulted in a four-fold enhancement over background of the TIE-2specific binding signal.

EXAMPLE 5 C2C12-ras CCM Contains an Activity that Induces TyrosinePhosphorylation of TIE-2 Receptor

C2C12-ras 10× CCM was examined for its ability to induce tyrosinephosphorylation of TIE-2 in ABAE cells. Serum-starved ABAE cells werebriefly incubated with C2C12-ras CCM, lysed and subjected toimmunoprecipitation and Western analyses as described above. Stimulationof serum-starved ABAE cells with serum-free C2C12-ras 10× CCM was doneas follows. The medium of ABAE cells starved as described above wasremoved and replaced with either defined medium or 10× CCM that had beenpre-warmed to 37° C. After 10 minutes, the media were removed and thecells were twice rinsed on ice with an excess of chilled PBSsupplemented with orthovanadate/NaF/benzamidine. Cell lysis andTIE-2-specific immunoprecipitation was done as described above.

ABAE cells incubated for 10 minutes with defined medium showed noinduction of TIE-2 tyrosine phosphorylation, whereas incubation withC2C12-ras CCM stimulated at least a 100× increase in TIE-2phosphorylation. This activity was almost totally depleted bypre-incubation of the C2C12-ras 10× CCM for 90 minutes at roomtemperature with 13 μg of TIE-2 RB coupled to protein G-Sepharose beads.Medium incubated with protein G Sepharose alone was not depleted of thisphosphorylating activity.

EXAMPLE 6 Expression Cloning of TIE-2 Ligand

COS-7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM)containing 10% fetal bovine serum (FBS), 1% each of penicillin andstreptomycin (P/S) and 2 mM glutamine in an atmosphere of 5% CO₂. Themouse myoblast C2C12 ras cell line was cultured in Eagle's minimalessential medium (EMEM) with 10% FBS, (P/S) and 2 mM glutamine. Fulllength mouse TIE-2 ligand cDNA clones were obtained by screening a C2C12ras cDNA library in the pJFE14 vector expressed in COS cells. Thisvector, as shown in FIG. 2, is a modified version of the vector pSR_(α)(Takebe, et al. 1988, Mol. Cell. Biol. 8:466-472). The library wascreated using the two BSTX1 restriction sites in the pJFE14 vector.

COS-7 cells were transiently transfected with either the pJFE14 libraryor control vector by the DEAE-dextran transfection protocol. Briefly,COS-7 cells were plated at a density of 1.0×10⁶ cells/100 mm plate 24hours prior to transfection. For transfection, the cells were culturedin serum-free DMEM containing 400 μg/ml of DEAE-dextran, 1 μMchloroquine, and 2 mM glutamine, and 1 μg of the appropriate DNA for 3-4hours at 37° C. in an atmosphere of 5% CO₂. The transfection media wasaspirated and replaced with PBS with 10% DMSO for 2-3 min. Followingthis DMSO “shock”, the COS-7 cells were placed into DMEM with 10% FBS,1% each of penicillin and streptomycin, and 2 mM glutamine for 48 hours.

Because the TIE-2 ligand is secreted it was necessary to permeabilizethe cells to detect binding of the receptorbody probe to the ligand. Twodays after transfection the cells were rinsed with PBS and thenincubated with PBS containing 1.8% formaldehyde for 15-30 min. at roomtemperature. Cells were then washed with PBS and incubated for 15 min.with PBS containing 0.1% Triton X-100 and 10% Bovine Calf Serum topermeabilize the cells and block non-specific binding sites.

The screening was conducted by direct localization of staining using aTIE-2 receptorbody (RB), which consisted of the extracellular domain ofTIE-2 fused to the IgG1 constant region. This receptorbody was preparedas set forth in Example 2. A 100 mm dish of transfected, fixed andpermeabilized COS cells was probed by incubating them for 30 min withTIE-2 RB. The cells were then washed twice with PBS and incubated for anadditional 30 min with PBS/10% Bovine Calf Serum/anti-human IgG-alkalinephosphatase conjugate. After three PBS washes, cells were incubated inalkaline-phosphatase substrate for 30-60 min. The dish was theninspected microscopically for the presence of stained cells. For eachstained cell, a small area of cells including the stained cell wasscraped from the dish using a plastic pipette tip and plasmid DNA wasthen rescued and used to electroporate bacterial cells. Single bacterialcolonies resulting from the electroporation were picked and plasmid DNAprepared from these colonies was used to transfect COS-7 cells whichwere probed for TIE-2 ligand expression as evidenced by binding to TIE-2receptorbodies. This allowed identification of single clones coding forTIE-2 ligand. Confirmation of TIE-2 ligand expression was obtained byphosphorylation of the TIE-2 receptor using the method set forth inExample 5. A plasmid clone encoding the TIE-2 ligand was deposited withthe ATCC on Oct. 7, 1994 and designated as “pJFE14 encoding TIE-2ligand” under ATCC Accession No. 75910.

EXAMPLE 7 Isolation and Sequencing of Full Length cDNA Clone EncodingHuman TIE-2 Ligand

A human fetal lung cDNA library in lambda gt-10 (see FIG. 3) wasobtained from Clontech Laboratories, Inc. (Palo Alto, Calif.). Plaqueswere plated at a density of 1.25×10⁶/20×20 cm plate, and replica filterstaken following standard procedures (Sambrook, et al., MolecularCloning: A Laboratory Manual, 2nd Ed., page 8.46, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.).

Isolation of human tie-2 ligand clones was carried out as follows. A 2.2kb Xhol fragment from the deposited tie-2 ligand clone (ATCC NO.75910—see Example 6 above) was labeled by random priming to a specificactivity of approximately 5×10⁸ cpm/ng. Hybridization was carried out at65° C. in hybridization solution containing 0.5 mg/ml salmon sperm DNA.The filters were washed at 65° C. in 2×SSC, 0.1% SDS and exposed toKodak XAR-5 film overnight at −70° C. Positive phage were plaquepurified. High titre phage lysates of pure phage were used for isolationof DNA via a Qiagen column using standard techniques (Qiagen, Inc.,Chatsworth, Calif., 1995 catalog, page 36). Phage DNA was digested withEcoRI to release the cloned cDNA fragment for subsequent subcloning. Alambda phage vector containing human tie-2 ligand DNA was deposited withthe ATCC on Oct. 26, 1994 under the designation λgt10 encoding htie-2ligand 1 (ATCC Accession No. 75928). Phage DNA may be subjected directlyto DNA sequence analysis by the dideoxy chain termination method(Sanger, et al., 1977, Proc. Natl. Acad. Sci. U.S.A. 74: 5463-5467).

Subcloning of the human tie-2 ligand DNA into a mammalian expressionvector may be accomplished as follows. The clone λgt10 encoding htie-2ligand 1 contains an EcoRI site located 490 base pairs downstream fromthe start of the coding sequence for the human TIE-2 ligand. The codingregion may be excised using unique restriction sites upstream anddownstream of the initiator and stop codons respectively. For example,an Spel site, located 70 bp 5′ to the initiator codon, and a Bpu1102i(also known as Blpl) site, located 265 bp 3′ to the stop codon, may beused to excise the complete coding region. This may then be subclonedinto the pJFE14 cloning vector, using the Xbal (compatible to the Speloverhang) and the Pstl sites (the Pstl and Bpu1102i sites are both madeblunt ended).

The coding region from the clone λgt10 encoding htie-2 ligand 1 wassequenced using the ABI 373A DNA sequencer and Taq Dideoxy TerminatorCycle Sequencing Kit (Applied Biosystems, Inc., Foster City, Calif.).The nucleotide and deduced amino acid sequence of human TIE-2 ligandfrom the clone λgt10 encoding htie-2 ligand 1 is shown in FIGS. 4A-4D(SEQ ID NO: 1 and SEQ ID NO: 2).

In addition, full length human tie-2 ligand cDNA clones were obtained byscreening a human glioblastoma T98G cDNA library in the pJFE14 vector.Clones encoding human TIE-2 ligand were identified by DNA hybridizationusing a 2.2 kb XhoI fragment from the deposited tie-2 ligand clone (ATCCNO. 75910) as a probe (see Example 6 above). The coding region wassequenced using the ABI 373A DNA sequencer and Taq Dideoxy TerminatorCycle Sequencing Kit (Applied Biosystems, Inc., Foster City, Calif.).This sequence was nearly identical to that of clone λgt10 encodinghtie-2 ligand 1. As shown in FIGS. 4A-4D (SEQ ID NO: 1 and SEQ ID NO:2), the clone λgt10 encoding htie-2 ligand 1 contains an additionalglycine residue which is encoded by nucleotides 1114-1116. The codingsequence of the T98G clone does not contain this glycine residue butotherwise is identical to the coding sequence of the clone λgt10encoding htie-2 ligand 1. FIGS. 5A-5D (SEQ ID NO: 3 and SEQ ID NO: 4)sets forth the nucleotide and deduced amino acid sequence of human TIE-2ligand from the T98G clone.

EXAMPLE 8 Isolation and Sequencing of Second Full Length cDNA Clone aEncoding Human TIE-2 Ligand

A human fetal lung cDNA library in lambda gt-10 (see FIG. 3) wasobtained from Clontech Laboratories, Inc. (Palo Alto, Calif.). Plaqueswere plated at a density of 1.25×10⁶/20×20 cm plate, and replica filterstaken following standard procedures (Sambrook, et al., MolecularCloning: A Laboratory Manual, 2nd Ed., page 8.46, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.). Duplicate filters were screenedat low stringency (2×SSC, 55° C.) with probes made to the human TIE-2ligand 1 sequence. One of the duplicate filters was probed with a 5′probe, encoding amino acids 25-265 of human TIE-2 ligand 1 as set forthin FIGS. 4A-4D (SEQ ID NO: 1 and SEQ ID NO: 2). The second duplicatefilter was probed with a 3′ probe, encoding amino acids 282-498 of humanTIE-2 ligand 1 sequence (see FIGS. 4A-4D (SEQ ID NO: 1 and SEQ ID NO:2). Both probes were hybridized at 55° C. in hybridization solutioncontaining 0.5 mg/ml salmon sperm DNA. Filters were washed in 2×SSC at55° C. and exposed overnight to X-ray film. In addition, duplicatefilters were also hybridized at normal stringency (2×SSC, 65° C.) to thefull length coding probe of mouse TIE-2 ligand 1 (F3-15, XhoI insert).Three positive clones were picked that fulfilled the following criteria:i. hybridization had not been seen to the full length (mouse) probe atnormal stringency, and ii. hybridization was seen at low stringency toboth 5′ and 3′ probes. EcoRI digestion of phage DNA obtained from theseclones indicated two independent clones with insert sizes ofapproximately 2.2 kb and approximately 1.8 kb. The 2.2 kb EcoRI insertwas subcloned into the EcoRI sites of both pBluescript KS (Stratagene)and a mammalian expression vector suitable for use in COS cells. Twoorientations were identified for the mammalian expression vector. The2.2 kb insert in pBluescript KS was deposited with the ATCC on Dec. 9,1994 and designated as pBluescript KS encoding human TIE 2 ligand 2. Thestart site of the TIE-2 ligand 2 coding sequence is approximately 355base pairs downstream of the pBluescript EcoRI site.

COS-7 cells were transiently transfected with either the expressionvector or control vector by the DEAE-dextran transfection protocol.Briefly, COS-7 cells were plated at a density of 1.0×10⁶ cells/100 mmplate 24 hours prior to transfection. For transfection, the cells werecultured in serum-free DMEM containing 400 μg/ml of DEAE-dextran, 1 μMchloroquine, and 2 mM glutamine, and 1 μg of the appropriate DNA for 3-4hours at 37° C. in an atmosphere of 5% CO₂. The transfection media wasaspirated and replaced with phosphate-buffered saline with 10% DMSO for2-3 min. Following this DMSO “shock”, the COS-7 cells were placed intoDMEM with 10% FBS, 1% each of penicillin and streptomycin, and 2 mMglutamine for 48 hours.

Because the TIE-2 ligand is secreted it was necessary to permeabilizethe cells to detect binding of the receptorbody probe to the ligand.Transfected COS-7 cells were plated at a density of 1.0×10⁶ cells/100 mmplate. The cells were rinsed with PBS and then incubated with PBScontaining 1.8% formaldehyde for 15-30 min. at room temperature. Cellswere then washed with PBS and incubated for 15 min. with PBS containing0.1% Triton X-100 and 10% Bovine Calf Serum to permeabilize the cellsand block non-specific binding sites. The screening was conducted bydirect localization of staining using a TIE-2 receptorbody, whichconsisted of the extracellular domain of TIE-2 fused to the IgG1constant region. This receptorbody was prepared as set forth in Example2. Transfected COS cells were probed by incubating them for 30 min withTIE-2 receptorbody. The cells were then washed twice with PBS, fixedwith methanol, and then incubated for an additional 30 min with PBS/10%Bovine Calf Serum/anti-human IgG-alkaline phosphatase conjugate. Afterthree PBS washes, cells were incubated in alkaline-phosphatase substratefor 30-60 min. The dish was then inspected microscopically for thepresence of stained cells. Cells expressing one orientation of theclone, but not the other orientation, were seen to bind the TIE-2receptorbody.

One of skill in the art will readily see that the described methods maybe used to further identify other related members of the TIE ligandfamily.

The coding region from the clone pBluescript KS encoding human TIE-2ligand 2 was sequenced using the ABI 373A DNA sequencer and Taq DideoxyTerminator Cycle Sequencing Kit (Applied Biosystems, Inc., Foster City.Calif.). The nucleotide and deduced amino acid sequence of human TIE-2ligand from the clone pBluescript KS encoding human TIE-2 ligand 2 isshown in FIGS. 6A-6D (SEQ ID NO: 5 and SEQ ID NO: 6).

EXAMPLE 9 TIE-2 Ligand 2 is a Receptor Antagonist

Conditioned media from COS cells expressing either TIE-2 ligand 2 (TL2)or TIE-2 ligand 1 (TL1) were compared for their ability to activateTIE-2 receptors naturally present in human endothelial cell lines.

Lipofectamine reagent (GIBCO-BRL, Inc.) and recommended protocols wereused to transfect COS-7 cells with either the pJFE14 expression vectoralone, pJFE14 vector containing the human TIE-2 ligand 1 cDNA, or with apMT21 expression vector (Kaufman, R. J., 1985, Proc. Natl. Acad. Sci.USA 82: 689-693) containing the human TIE-2 ligand 2 cDNA. COS mediacontaining secreted ligands were harvested after three days andconcentrated 20-fold by diafiltration (DIAFLO ultrafiltration membranes,Amicon, Inc.). The quantity of active TIE-2 ligand 1 and TIE-2 ligand 2present in these media was determined and expressed as the amount (inresonance units, R.U.) of TIE-2 receptor specific binding activitymeasured by a BIAcore binding assay.

Northern (RNA) analyses revealed significant levels of TIE-2 transcriptsin HAEC (Human Aortic Endothelial Cell) human primary endothelial cells(Clonetics, Inc.). Therefore, these cells were used to examine whetherTIE-2 receptor is tyrosine-phosphorylated when exposed to COS mediacontaining the TIE-2 ligands. HAEC cells were maintained in a completeendothelial cell growth medium (Clonetics, Inc.) that contained 5% fetalbovine serum, soluble bovine brain extract, 10 ng/ml human EGF, 1 mg/mlhydrocortisone, 50 mg/ml gentamicin and 50 ng/ml amphotericin-B.Assessment of whether TL1 and TL2 could activate TIE-2 receptor in theHAEC cells was done as follows. Semi-confluent HAEC cells wereserum-starved for two hours in high-glucose Dulbecco's MEM with addedL-glutamine and penicillin-streptomycin at 37° C. followed byreplacement of the starvation medium with ligand-containing conditionedCOS media for 7 minutes at 37° C. in a 5% CO2 incubator. The cells weresubsequently lysed and TIE-2 receptor protein was recovered byimmunoprecipitation of the lysates with TIE-2 peptide antiserum,followed by Western blotting with antiphosphotyrosine antiserum, exactlyas described in example 1. The results are shown in FIG. 7.Phosphotyrosine levels on the TIE-2 receptor (TIE-2-R) were induced bytreatment of HEAC cells with TIE-2 ligand 1 (Lane L1) but not by TIE-2ligand 2 (Lane L2) conditioned COS media. MOCK is conditioned media fromCOS transfected with JFE14 empty vector.

Evidence that both TL1 and TL2 specifically bind to the TIE-2 receptorwas demonstrated by using a BIAcore to assay the TIE-2 receptor specificbinding activities in transfected COS media and by immunostaining ofTL1- and TL2-expressing COS cells with TIE-2 receptorbodies.

Because TL2 did not activate the TIE-2 receptor, applicants set out todetermine whether TL2 might be capable of serving as an antagonist ofTL1 activity. HAEC phosphorylation assays were performed in which cellswere first incubated with an “excess” of TL2, followed by addition ofdilute TL1. It was reasoned that prior occupancy of TIE-2 receptor dueto high levels of TL2 might prevent subsequent stimulation of thereceptor following exposure to TL1 present at a limiting concentration.

Semi-confluent HAEC cells were serum-starved as described above and thenincubated for 3 min., at 37° C. with 1-2 ml. of 20× COS/JFE14-TL2conditioned medium. Control plates were treated with 20× COS/JFE14-onlymedium (MOCK). The plates were removed from the incubator and variousdilutions of COS/JFE14-TL1 medium were then added, followed by furtherincubation of the plates for 5-7 min. at 37° C. Cells were subsequentlyrinsed, lysed and TIE-2-specific tyrosine phosphorylation in the lysateswas examined by receptor immunoprecipitation and Western blotting, asdescribed above. TL1 dilutions were made using 20× COS/JFE14-TL1 mediumdiluted to 2×, 0.5×, 0.1×, or 0.02× by addition of 20× COS/JFE14-alonemedium. An assay of the initial 20× TL1 and 20× TL2 COS media usingBIAcore biosensor technology indicated that they contained similaramounts of TIE-2-specific binding activities, i.e., 445 R.U. and 511R.U. for TL1 and TL2, respectively. The results of theantiphosphotyrosine Western blot, shown in FIG. 8, indicate that whencompared to prior treatment of HAEC cells with MOCK medium (lane 1),prior treatment of HAEC cells with excess TIE-2 ligand 2 (lane 2)antagonizes the subsequent ability of dilute TIE-2 ligand 1 to activatethe TIE-2 receptor (TIE-2-R).

The ability of TL2 to competitively inhibit TL1 activation of theTIE-2-R was further demonstrated using the human cell hybrid line,EA.hy926 (see Example 21 for detailed description of this cell line andits maintenance). Experiments were performed in which unconcentrated COScell media containing TL1 were mixed at varying dilutions with eitherMOCK- or TL2-conditioned media and placed on serum-starved EA.hy926 cellmonolayers for 5 minutes at 37° C. The media were then removed, thecells were harvested by lysis and TIE-2-specific tyrosinephosphorylation was examined by Western blots, as described above. FIG.9 shows an experiment which contains three groups of treatments, asviewed from left to right. As shown in the four lanes at the left,treatment of the EA.hy926 cells with 1× COS-TL1 alone robustly activatedthe endogenous TIE-2-R in these cells, whereas 1× TL2 COS medium wasinactive. However, mixture of TL1 with either MOCK or TL2 demonstratedthat TL2 can block the activity of TL1 in a dose-dependent fashion. Inthe central three pairs of lanes the ratio of TL2 (or MOCK) wasdecreased while the amount of TL1 in the mixture was correspondinglyincreased from 0.1× to 0.3×. At any of these mixture ratios the TL1:TL2lanes showed a reduced level of TIE-2-R phosphorylation compared to thatof the corresponding TL1:MOCK lanes. When the amount TL1 was held steadyand the amount of TL2 (or MOCK) was decreased, however (shown in thethree pairs of lanes at the right), a point was reached at which the TL2in the sample was too dilute to effectively inhibit TL1 activity. Therelative amount of each ligand present in these conditioned COS mediacould be estimated from their binding units as measured by the BIAcoreassay and from Western blots of the COS media with ligand-specificantibodies. Consequently, we can infer that only a few-fold molar excessof TL2 is required to effectively block the activity of TL1 in vitro.This is significant because we have observed distinct examples in vivo(see Example 17 and FIG. 16) where TL2 mRNAs achieve considerableabundance relative to those of TL1. Thus, TL2 may be serving animportant physiological role in effectively blocking signaling by theTIE-2-R at these sites.

Taken together these data confirm that, unlike TL1, TL2 is unable tostimulate endogenously expressed TIE-2-R on endothelial cells.Furthermore, at a few fold molar excess TL2 can block TL1 stimulation ofthe TIE-2 receptor, indicating that TL2 is a naturally occurring TIE-2receptor antagonist.

EXAMPLE 10 Identification of TIE-2-Specific Binding Activity inConditioned Medium and COS Cell Supernatants

Binding activity of 10× CCM from the cell lines C2C12-ras, Rat2 ras,SHEP, and T98G, or COS cell supernatants after transfection with eitherhuman TIE-2 ligand 1 (hTL1) or human TIE-2 ligand 2 (hTL2) was measuredusing biosensor technology (BIAcore; Pharmacia Biosensor, Piscataway,N.J.) which monitors biomolecular interactions in real-time via surfaceplasmon resonance (SPR). Purified rat or human TIE-2 RB was covalentlycoupled through primary amines to the carboxymethyl dextran layer of aCM5 research grade sensor chip (Pharmacia Biosensor; Piscataway, N.J.).The sensor chip surface was activated using a mixture ofN-hydroxysuccinimide (NHS) andN-ethyl-N′-(3-dimethylaminopropyl)carbodiimide (EDC), followed byimmobilization of TIE-2 RB (25 μg/mL, pH 4.5) and deactivation ofunreacted sites with 1.0 M ethanolamine (pH 8.5). In general, 9000-10000RU of each receptorbody was coupled to the sensor chip.

The running buffer used in the system was HBS (10 mM Hepes, 150 mM NaCl,0.005% P20 surfactant, pH 7.4). The samples were centrifuged for 15 minat 4° C. and further clarified using a sterile, low protein-binding 0.45μm filter (Millipore; Bedford, Mass.). Dextran (2 mg/ml) and P20surfactant (0.005%) were added to each sample. Aliquots of 40 μL wereinjected across the immobilized surface (either rat or human TIE-2) at aflow rate of 5 μL/min and the receptor binding was monitored for 8 min.The binding activity (resonance units, RU) was measured as thedifference between a baseline value determined 30 s prior to the sampleinjection and a measurement taken at 30 s post-injection. Regenerationof the surface was accomplished with one 15-μL pulse of 3 M MgCl₂.

The CCM samples (C2C12-ras, Rat2-ras, SHEP, T98G) were tested on the ratTIE-2 RB immobilized surface, while the recombinant hTL1 and hTL2 weretested on the human TIE-2 RB immobilized surface. In each case, specificbinding to the TIE-2 receptorbody was evaluated by incubating thesamples with 25 μg/ml of either soluble TIE-2 (rat or human) RB or trkBRB prior to assaying the binding activity. As shown in FIGS. 10A-10D andFIGS. 11A-11B, the addition of soluble trkB RB causes a slight decreasein the TIE-2 binding activity, while the addition of soluble TIE-2 RBsignificantly reduces the binding activity as compared to that measuredin the absence of TIE-2 RB.

EXAMPLE 11 TIE-2 RB Specifically Blocks Activation of the TIE-2 Receptorby TIE-2 Ligand 1

The applicants sought to determine whether soluble TIE-2 RB can serve asa competitive inhibitor to block activation of TIE-2 receptor by TIE-2ligand 1 (TL1). To do this, TL1-containing COS media were preincubatedwith either TIE-2- or TrkB-RB and then compared for their ability toactivate TIE-2 receptors naturally present in a human endothelial cellline.

Conditioned COS media were generated from COS-7 cells transfected witheither the pJFE14 expression vector alone (MOCK), or pJFE14 vectorcontaining the human TIE-2 ligand 1 cDNA (TL1) and harvested asdescribed in Example 9 hereinabove, with the exception that the mediawere sterile filtered but not concentrated. The quantity of TL1 wasdetermined and expressed as the amount (in resonance units, R.U.) ofTIE-2 receptor-specific binding activity measured by BIAcore bindingassay.

Northern (RNA) analyses revealed significant levels of tie-2 transcriptsin HUVEC (Human Umbilical Vein Endothelial Cell) human primaryendothelial cells (Clonetics, Inc.). Therefore, these cells were used toexamine whether TIE-2 receptor can be tyrosine-phosphorylated whenexposed in the presence of TIE-2- or TrkB-RBs to COS media containingTL1. HUVEC cells were maintained at 37° C., 5% CO₂ in a completeendothelial cell growth medium (Clonetics, Inc.) that contained 5% fetalbovine serum, soluble bovine brain extract with 10 μg/ml heparin, 10ng/ml human EGF, 1 ug/ml hydrocortisone, 50 μg/ml gentamicin and 50ng/ml amphotericin-B. Assessment of whether TL1 could activate TIE-2receptor in the HUVEC cells was done as follows. Confluent dishes ofHUVEC cells were serum-starved for two-to-four hours in low-glucoseDulbecco's MEM at 37° C., 5% CO₂, followed by 10 minute incubation instarvation medium that included 0.1 mM sodium orthovanadate, a potentinhibitor of phosphotyrosine phosphatases. Meanwhile, conditioned COSmedia were preincubated 30 min. at room temperature with either TIE-2-or TrkB-RB added to 50 μg/ml. The starvation medium was then removedfrom the HUVEC dishes and incubated with the RB-containing COS media for7 minutes at 37° C. HUVEC cells were subsequently lysed and TIE-2receptor protein was recovered by immunoprecipitation with TIE-2 peptideantiserum, followed by Western blotting with an anti-phosphotyrosineantibody, as described in Example 1.

The results are shown in FIG. 12. Phosphotyrosine levels on the TIE-2receptor were induced by treatment of HUVEC cells with TIE-2 ligand 1(TL1) relative to that seen with control medium (MOCK) and thisinduction is specifically blocked by prior incubation with TIE-2-RB(TIE-2-Fc) but not by incubation with TrkB-RB (TrkB-Fc). These dataindicate that soluble TIE-2 RB can serve as a selective inhibitor toblock activation of TIE-2 receptor by TIE-2 ligand 1.

EXAMPLE 12 Construction of TIE-2 Ligandbodies

An expression construct was created that would yield a secreted proteinconsisting of the entire coding sequence of human TIE-2 ligand 1 (TL1)or TIE-2 ligand 2 (TL2) fused to the human immunoglobulin gamma-1constant region (IgG1 Fc). These fusion proteins are called TIE-2“ligandbodies” (TL1-Fc or TL2-Fc). The Fc portion of TL1-Fc and TL2-Fcwas prepared as follows. A DNA fragment encoding the Fc portion of humanIgG1 that spans from the hinge region to the carboxy-terminus of theprotein, was amplified from human placental cDNA by PCR witholigonucleotides corresponding to the published sequence of human IgG1;the resulting DNA fragment was cloned in a plasmid vector. AppropriateDNA restriction fragments from a plasmid encoding full-length TL1 or TL2and from the human IgG1 Fc plasmid were ligated on either side of ashort PCR-derived fragment that was designed so as to fuse, in-frame,TL1 or TL2 with human IgG1 Fc protein-coding sequences.

Milligram quantities of TL2-Fc were obtained by cloning the TL2-Fc DNAfragment into the pVL1393 baculovirus vector and subsequently infectingthe Spodoptera frugiperda SF-21AE insect cell line. Alternatively, thecell line SF-9 (ATCC Accession No. CRL-1711) or the cell lineBTI-TN-5b1-4 may be used. DNA encoding the TL2-Fc was cloned as an EcoRI-NotI fragment into the baculovirus transfer plasmid pVL1393. PlasmidDNA was recombined into viral DNA by mixing 3 μg of plasmid DNA with 0.5μg of Baculo-Gold DNA (Pharminigen), followed by introduction intoliposomes using 30 μg Lipofectin (GIBCO-BRL). DNA-liposome mixtures wereadded to SF-21AE cells (2×106 cells/60 mm dish) in TMN-FH medium(Modified Grace's Insect Cell Medium (GIBCO-BRL) for 5 hours at 27° C.,followed by incubation at 27° C. for 5 days in TMN-FH mediumsupplemented with 5% fetal calf serum. Tissue culture medium washarvested for plaque purification of recombinant viruses, which wascarried out using methods previously described (O'Reilly, D. R., L. K.Miller, and V. A. Luckow, Baculovirus Expression Vectors—A LaboratoryManual. 1992, New York: W. H. Freeman) except that the agarose overlaycontained 125 mg/mL X-gal(5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside; GIBCO-BRL). After 5days of incubation at 27° C., non-recombinant plaques were scored bypositive chromogenic reaction to the X-gal substrate, and theirpositions marked. Recombinant plaques were then visualized by additionof a second overlay containing 100 mg/mL MTT(3-[4,5-dimethylthiazol-2-yl]2,5,diphenyltetrazolium bromide; Sigma).Putative recombinant virus plaques were picked by plug aspiration, andpurified by multiple rounds of plaque isolation to assure homogeneity.Virus stocks were generated by serial, low-multiplicity passage ofplaque-purified virus. Low passage stocks of one virus clone (vTL2-FcClone #7) were produced.

SF-21AE cells were cultured in serum-free medium (SF-900 II, Gibco BRL)containing 1× antibiotic/antimycotic solution (Gibco BRL) and 25 mg/LGentamycin (Gibco BRL). Pluronic F-68 was added as a surfactant to afinal concentration of 1 g/L. Cultures (4 L) were raised in a bioreactor(Artisan Cell Station System) for at least three days prior toinfection. Cells were grown at 27° C., with gassing to 50% dissolvedoxygen, at a gas flow rate of 80 mL/min (aeration at a sparge ring).Agitation was by means of a marine impeller at a rate of 100 rpm. Cellswere harvested in mid-logarithmic growth phase (˜2×10 6 cells/mL),concentrated by centrifugation, and infected with 5 plaque forming unitsof vTL2-Fc per cell. Cells and inoculum were brought to 400 mL withfresh medium, and virus was adsorbed for 2 hours at 27° C. in a spinnerflask. The culture was then resuspended in a final volume of 8 L withfresh serum-free medium, and the cells incubated in the bioreactor usingthe previously described conditions.

Culture medium from vTL2-Fc-infected SF21AE cells were collected bycentrifugation (500×g, 10 minutes) at 72 hours post-infection. Cellsupernatants were brought to pH 8 with NaOH. EDTA was added to a finalconcentration of 10 mM and the supernatant pH was readjusted to 8.Supernatants were filtered (0.45 μm, Millipore) and loaded on a proteinA column (protein A sepharose 4 fast flow or HiTrap protein A, both fromPharmacia). The column was washed with PBS containing 0.5 M NaCl untilthe absorbance at 280 nm decreased to baseline. The column was washed inPBS and eluted with 0.5 M acetic acid. Column fractions were immediatelyneutralized by eluting into tubes containing 1 M Tris pH 9. The peakfractions containing the TL2-Fc were pooled and dialyzed versus PBS.

EXAMPLE 13 Expression of TIE-1, TIE-2, TL1, and TL2 in Renal CellCarcinoma

In situ hybridization experiments were performed on human renal cellcarcinoma tumor tissue using TIE-1, TIE-2, TL1, and TL2 cDNA probes.TIE-2, TIE-1, TL1, and TL2 expression were all up-regulated in the tumorvasculature. Ligand expression appeared to be localized to either thevascular endothelial cells (TL2) or very near the vascular endothelialcells in the mesenchyme (TL1). VEGF has been shown to be dramaticallyup-regulated in this tumor tissue. Brown, et al. Am. J. Pathol.143:1255-1262 (1993).

EXAMPLE 14 Expression of TIE-1, TIE-2, TL1, and TL2 in Wound Healing

In situ hybridization experiments were performed on cross-sectionaltissue slices obtained from a rat cutaneous wound model using TIE-1,TIE-2, TL1, and TL2 cDNA probes. The wound healing model involvespressing a small cork bore against the skin of a rat and removing asmall, cylindrical plug of skin. As healing begins at the base of thewound, a vertical slice of tissue is taken and used for in situhybridization. In the tested tissue sample, TL1 and TL2 appeared to beslightly up-regulated by four days post-injury. In contrast to theslightly up-regulated expression of TL1 and TL2 in this tissue, VEGFexpression, which may precede TL1 and TL2 expression, is dramaticallyup-regulated.

EXAMPLE 15 Expression of TIE Ligands in Fetal Liver and Thymus

Reverse transcription-PCR (RT-PCR) was performed on mouse E14.5 fetalliver and mouse E17.5 fetal thymus. Agarose gel electrophoresis of theRT-PCR products revealed that in the mouse fetal liver, TIE-2 ligand 1(TL1) RNA is enriched in the stromal region, but is absent inc-kit⁺TER119 hematopoietic precursor cells. In this same tissue, TIE-2ligand 2 (TL2) RNA is enriched in the stromal cells, but absent in thehematopoietic precursor cells (FIG. 13). In the mouse fetal thymus, TL2is enriched in the stromal cells (FIG. 14).

EXAMPLE 16 The TIE Receptor/Ligand System in Angiogenesis

Although the TIE-2/TIE ligand system appears to play an important rolein endothelial cell biology, it has not been shown to play asignificant, active role in the early to intermediate stages ofvascularization (f angioblast or endothelial cell proliferation andmigration, tubule formation, and other early stage events in vascularmodeling). In contrast to the receptors and factors known to mediatethese aspects of vascular development, the temporally late pattern ofexpression of TIE-2 and TL1 in the course of vascularization suggeststhat this system plays a distinct role in the latter stages vasculardevelopment, including the structural and functional differentiation andstabilization of new blood vessels. The pattern of expression ofTIE-2/TL1 also is consistent with a continuing role in the maintenanceof the structural integrity and/or physiological characteristics of anestablished vasculature.

TIE Ligand 2 (TL2) appears to be a competitive inhibitor of TL1. Thespatiotemporal characteristics of TL2 expression suggest that thissingle inhibitory molecule may play multiple, context-dependent rolesessential to appropriate vascular development or remodeling (e.g.de-stabilization/de-differentiation of mature endothelial cells allowingthe formation of new vessels from existing vasculature, inhibition ofinappropriate blood vessel formation, and regression/involution ofmature blood vessels). FIG. 15 is a schematic representation of thehypothesized role of the TIE-2/TIE ligands in angiogenesis. In thisfigure TL1 is represented by (•), TL2 is represented by (*), TIE-2 isrepresented by (T), VEGF is represented by ([ ]), and flk-1 (a VEGFreceptor) is represented by (Y).

EXAMPLE 17 Expression of TIE Ligands in the Female Reproductive System:Expression in the Ovary

Preliminary observations made in experiments examining the expression ofthe TIE receptors and ligands in the female reproductive system areconsistent with the hypothesis the TL1 plays a role inneovascularization which temporally follows that of VEGF. The pattern ofTL2 expression is also consistent with an antagonism of the action ofTL1, and a specific role in vascular regression. To verify this,expression of relevant mRNAs can be examined following experimentalinduction of follicular and luteal development so that their temporalrelation to various aspects of neovascularization/vascular regressioncan be more clearly defined (e g in conjunction with endothelial cellstaining, vascular fills). Angiogenesis associated with folliculardevelopment and corpus luteum formation in staged ovaries of mature,female rats or following induced ovulation in pre-pubertal animals wasfollowed using in situ hybridization. FIG. 16 contains photographs of insitu hybridization slides showing the temporal expression pattern ofTIE-2, TL1, TL2, and VEGF during the ovarian cycle [Column 1: Earlypre-ovulatory follicle; Column 2: pre-ovulatory follicle; Column 3:early corpus luteum; and Column 4: atretic follicle; Row A:bright field;Row B:VEGF; Row C: TL2;

Row D: TL1 and Row E: TIE-2 receptor]. These studies revealed that VEGF,TL1 and TL2 are expressed in a temporally and spatially coordinatefashion with respect to the development and regression of vasculature inthe ovary, specifically with respect to the establishment of thevascular system which is generated in the course of the conversion of anovarian follicle to a corpus luteum (CL).

Briefly, VEGF expression increases in the follicular granule layer priorto its vascularization during the process of luteinization. During theprocess of CL formation, highest levels of VEGF expression are apparentin the center of the developing CL in the vicinity of luteinizing cellswhich are not yet vascularized. VEGF levels remain moderately high andare diffusely distributed in the developed CL. In contrast, noticeablyenhanced expression of TIE-2 ligand 1 occurs only late in process of CLformation, after a primary vascular plexus has been established. Later,TL1 expression is apparent throughout the CL at which time thedefinitive capillary network of the CL has been established.

TL2 exhibits a more complex pattern of expression than either VEGF orTL1. In the developing CL, TL2 is expressed at highest levels at thefront of the developing capillary plexus between the central avascularregion of the CL where VEGF expression is highest, and the mostperipheral portion of the CL where TL1 expression is dominant and wherethe luteinization process is complete and the vascular system is mostmature. TL2 also appears to be expressed at high levels in thefollicular layer of large follicles which are undergoing atresia. WhileTL1 is also apparent in atretic follicles, VEGF is not expressed.

The pattern of expression described above is most consistent with a rolefor VEGF in the initiation of angiogenesis, with TL1 acting late in thisprocess-for example in modeling and/or stabilization of the definitivevascular network. In contrast, TL2 is present both in areas of activeexpansion of a newly forming vascular network (during CL formation), andin regions which fail to establish a new vasculature and vascularregression is in progress (atretic follicles). This suggests a moredynamic and complex role for TL2, possibly involving destabilization ofexisting vasculature (necessary for regression) or developingvasculature (necessary for the dynamic modeling of newly formingvessels).

EXAMPLE 18 A Receptorbody Binding Assay and a Ligand Binding andCompetition Assay

A quantitative cell-free binding assay with two alternate formats hasbeen developed for detecting either TIE-2 receptorbody binding or ligandbinding and competition. In the receptorbody binding version of theassay, TIE-2 ligands (purified or partially purified; either TL1 or TL2)are coated onto an ELISA plate. Receptorbody at varying concentrationsis then added, which binds to the immobilized ligand in a dose-dependentmanner. At the end of 2 hours, excess receptorbody is washed away, thenthe amount bound to the plate is reported using a specific anti-human Fcantibody which is alkaline phosphatase tagged. Excess reporter antibodyis washed away, then the AP reaction is developed using a coloredsubstrate. The assay is quantitated using a spectrophotometer. FIG. 19shows a typical TIE-2-IgG binding curve. This assay has been used toevaluate the integrity of TIE-2-IgG after injection into rats and mice.The assay can also be used in this format as a ligand competition assay,in which purified or partially-purified TIE ligands compete withimmobilized ligand for receptorbody. In the ligand binding andcompetition version of the binding assay, TIE-2 ectodomain is coatedonto the ELISA plate. The Fc-tagged fibrinogen-like domain fragments ofthe TIE ligands (TL1-fFc and TL2-fFc) then bind to the ectodomain, andcan be detected using the same anti-human Fc antibody as describedabove. FIG. 20 shows an example of TL1-fFc binding to TIE-2 ectodomain.This version of the assay can also be used to quantitate levels ofTL1-fFc in serum or other samples. If untagged ligand (again, eitherpurified or unpurified) is added at the same time as the TL1-fFc, then acompetition is set up between tagged ligand fragment and full-lengthligand. The full-length ligand can displace the Fc-tagged fragment, anda competition curve is generated.

EXAMPLE 19 EA.hy926 Cell Line can be Used as a Reporter Cell Line forTIE Ligand Activity

EA.hy926 is a cell hybrid line that was established by fusion of HUVECwith the human lung carcinoma-derived line, A549 [Edgell, et al. Proc.Natl. Acad. Sci. (USA) 80, 3734-3737 (1983). EA.hy926 cells have beenfound to express significant levels of TIE-2 receptor protein with lowbasal phosphotyrosine levels. The density at which EA.hy926 cells arepassaged prior to their use for receptor assays, as well as their degreeof confluency at the time of assay, can affect TIE-2 receptor abundanceand relative inducibility in response to treatment with ligand. Byadopting the following regimen for growing these cells the EA.hy926 cellline can be used as a dependable system for assay of TIE-2 ligandactivities.

EA.hy926 cells are seeded at 1.5×10⁶ cells in T-75 flasks (Falconware)and re-fed every other day with high-glucose Dulbecco's MEM, 10% fetalbovine serum, L-glutamine, penicillin-streptomycin, and 1×hypoxanthine-aminopterin-thymidine (HAT, Gibco/BRL). After three to fourdays of growth, the cells are passaged once again at 1.5×10⁶ cells perT-75 flask and cultured an additional three to four days. Forphosphorylation assays, cells prepared as described above wereserum-starved by replacement of the culture medium with high-glucoseDMEM and incubation for 2-3 hours at 37° C. This medium was aspiratedfrom the flask and samples of conditioned media or purified ligand wereadded to the flask in a total volume of 1.5 ml followed by incubation at37° C. for 5 minutes. Flasks were removed from the incubator and placedon a bed of ice. The medium was removed and replaced with 1.25 ml LysisBuffer containing 1% nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS in20 mM Tris, pH 7.6, 150 mM NaCl, 50 mM NaF, 1 mM sodium orthovanadate, 5mM benzamidine, and 1 mM EDTA containing the protease inhibitors PMSF,aprotinin, and leupeptin. After 10 minutes on ice to allow membranesolubilization, plates were scraped and cell lysates were clarified bymicrocentrifugation at top speed for 10 minutes at 4° C. TIE-2 receptorwas immunoprecipitated from the clarified supernatant by incubation inthe cold with an anti-TIE-2 polyclonal antiserum and ProteinG-conjugated Sepharose beads. The beads were washed three times withcold cell lysis buffer and boiled 5 minutes in Laemmli sample buffer,which was then loaded on 7.5% SDS-polyacrylamide gels. Resolved proteinswere electrotransferred to PVDF (Lamblia-P) membrane and then subjectedto Western blot analysis using anti-phosphotyrosine antibody and the ECLreagent. Subsequent comparison of total TIE-2 protein levels on the sameblots was done by stripping the anti-phosphotyrosine antibody andreincubating with a polyclonal antiserum specific to the ectodomain ofTIE-2.

EXAMPLE 20 Isolation and Sequencing of Full Length cDNA Clone EncodingMammalian TIE Ligand-3

TIE ligand-3 (TL3) was cloned from a mouse BAC genomic library (ResearchGenetics) by hybridizing library duplicates, with either mouse TL1 ormouse TL2 probes corresponding to the entire coding sequence of thosegenes. Each copy of the library was hybridized using phosphate buffer at55° C. overnight. After hybridization, the filters were washed using2×SSC, 0.1% SDS at 60° C., followed by exposure of X ray film to thefilters. Strong hybridization signals were identified corresponding tomouse TL1 and mouse TL2. In addition, signals were identified whichweakly hybridized to both mouse TL1 and mouse TL2. DNA corresponding tothese clones was purified, then digested with restriction enzymes, andtwo fragments which hybridized to the original probes were subclonedinto a bacterial plasmid and sequenced. The sequence of the fragmentscontained two exons with homology to both mouse TL1 and mouse TL2.Primers specific for these sequences were used as PCR primers toidentify tissues containing transcripts corresponding to TL3. A PCR bandcorresponding to TL3 was identified in a mouse uterus cDNA library inlambda gt-11. (Clontech Laboratories, Inc., Palo Alto, Calif.).

Plaques were plated at a density of 1.25×10⁶/20×20 cm plate and replicafilters taken following standard procedures (Sambrook, et al., MolecularCloning: A Laboratory Manual, 2nd Ed., page 8.46, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.). Duplicate filters were screenedat “normal” stringency (2×SSC, 65° C.) with a 200 bp PCR radioactiveprobe made to the mouse TL3 sequence. Hybridization was at 65° C. in asolution containing 0.5 mg/ml salmon sperm DNA. Filters were washed in2×SSC at 65° C. and exposed for 6 hours to X-ray film. Two positiveclones that hybridized in duplicate were picked. EcoRI digestion ofphage DNA obtained from these clones indicated two independent cloneswith insert sizes of approximately 1.2 kb and approximately 2.2 kb. The2.2 kb EcoRI insert was subcloned into the EcoRI site of pBluescript KS(Stratagene). Sequence analysis showed that the longer clone was lackingan initiator methionine and signal peptide but otherwise encoded a probehomologous to both mouse TL1 and mouse TL2.

Two TL3-specific PCR primers were then synthesised as follows: US2:cctctgggctcgccagtttgttagg (SEQ ID NO: 29) US1: ccagctggcagatatcagg (SEQID NO: 30)

The following PCR reactions were performed using expression librariesderived from the mouse cell lines C2C12ras and MG87. In the primary PCRreaction, the specific primer US2 was used in conjunction withvector-specific oligos to allow amplification in either orientation. PCRwas in a total volume of 100 ml using 35 cycles of 94° C., 1 mm; 42° C.or 48° C. for 1 mm; 72° C, 1 mm. The secondary PCR reaction included thesecond specific primer, US1, which is contained within the primary PCRproduct, in conjunction with the same vector oligos. The secondaryreactions were for 30 cycles, using the same temperatures and times asprevious. PCR products were gel isolated and submitted for sequenceanalysis. On the basis of sequences obtained from a total of fourindependent PCR reactions using two different cDNA libraries, the 5′ endof the TL3 sequence was deduced. Northern analysis revealed moderate tolow levels of mouse TL3 transcript in mouse placenta. The expression ofmouse TL3 consisted of a transcript of approximately 3 kb. The fulllength TL3 coding sequence is set forth in FIGS. 21A-21C (SEQ ID NO: 9and SEQ ID NO: 10).

The mouse TL3 sequence may then be used to obtain a human clonecontaining the coding sequence of human TL3 by hybridizing either ahuman genomic or cDNA library with a probe corresponding to mouse TL3 ashas been described previously, for example, in Example 8 supra.

EXAMPLE 21 Isolation of Full Length Genomic Clone Encoding Human TIELigand-4

TIE ligand-4 (TL4) was cloned from a mouse BAC genomic library (BACHUMAN (II), Genome Systems Inc.) by hybridizing library duplicates, witheither a human TL1 radioactive probe corresponding to the entirefibrinogen coding sequence of TL1 (nucleotides 1153 to 1806 of FIGS.4A-4D [SEQ ID NO: 1 and SEQ ID NO: 2]) or a mouse TL3 radioactive probecorresponding to a segment of 186 nucleotides from the fibrinogen regionof mouse TL3 (nucleotides 1307 to 1492 of FIGS. 21A-21C [SEQ ID NO: 9and SEQ ID NO: 10]). Each probe was labeled by PCR using exactoligonucleotides and standard PCR conditions, except that dCTP wasreplaced by P³²dCTP. The PCR mixture was then passed through a gelfiltration column to separate the probe from free P³² dCTP. Each copy ofthe library was hybridized using phosphate buffer, and radioactive probeat 55° C. overnight using standard hybridization conditions. Afterhybridization, the filters were washed using 2×SSC, 0.1% SDS at 55° C.,followed by exposure of X ray film. Strong hybridization signals wereobserved corresponding to human TL1. In addition, signals wereidentified which weakly hybridized to both human TL1 and mouse TL3. DNAcorresponding to these clones was purified using standard procedures,then digested with restriction enzymes, and one fragment whichhybridized to the original probes was subcloned into a bacterial plasmidand sequenced. The sequence of the fragments contained one exon withhomology to both human TL1 and mouse TL3 and other members of the TIEligand family. Primers specific for these sequences may be used as PCRprimers to identify tissues containing transcripts corresponding to TL4.

The complete sequence of human TL4 may be obtained by sequencing thefull BAC clone contained in the deposited bacterial cells. Exons may beidentified by homology to known members of the TIE-ligand family such asTL1, TL2 and TL3. The full coding sequence of TL4 may then be determinedby splicing together the exons from the TL4 genomic clone which, inturn, may be used to produce the TL4 protein. Alternatively, the exonsmay be used as probes to obtain a full length cDNA clone, which may thenbe used to produce the TL4 protein. Exons may also be identified fromthe BAC clone sequence by homology to protein domains such as fibrinogendomains, coiled coil domains, or protein signals such as signal peptidesequences. Missing exons from the BAC clone m,ay be obtained byidentification of contiguous BAC clones, for example, by using the endsof the deposited BAC clone as probes to screen a human genomic librarysuch as the one used herein, by using the exon sequence contained in theBAC clone to screen a cDNA library, or by performing either 5′ or 3′RACE procedure using oligonucleotide primers based on the TL4 exonsequences.

Identification of Additional TIE Ligand Family Members

The novel TIE ligand-4 sequence may be used in a rational search foradditional members of the TIE ligand family using an approach that takesadvantage of the existence of conserved segments of strong homologybetween the known family members. For example, an alignment of the aminoacid sequences of the TIE ligands shows several regions of conservedsequence (see boxed regions of FIGS. 22A-22B (SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16).Degenerate oligonucleotides essentially based on these boxes incombination with either previously known or novel TIE ligand homologysegments may be used to identify new TIE ligands.

The highly conserved regions among TL1, TL2 and TL3 may be used indesigning degenerate oligonucleotide primers with which to prime PCRreactions using cDNAs. cDNA templates may be generated by reversetranscription of tissue RNAs using oligo d(T) or other appropriateprimers. Aliquots of the PCR reactions may then be subjected toelectrophoresis on an agarose gel. Resulting amplified DNA fragments maybe cloned by insertion into plasmids, sequenced and the DNA sequencescompared with those of all known TIE ligands.

Size-selected amplified DNA fragments from these PCR reactions may becloned into plasmids, introduced into E. coli by electroporation, andtransformants plated on selective agar. Bacterial colonies from PCRtransformation may be analyzed by sequencing of plasmid DNAs that arepurified by standard plasmid procedures.

Cloned fragments containing a segment of a novel TIE ligand may be usedas hybridization probes to obtain full length cDNA clones from a cDNAlibrary. For example, the human TL4 genomic sequence may be used toobtain a human cDNA clone containing the complete coding sequence ofhuman TL4 by hybridizing a human cDNA library with a probe correspondingto human TL4 as has been described previously.

EXAMPLE 22 Cloning of the Full Coding Sequence of hTL4

Both 5′ and 3′ coding sequence from the genomic human TL-4 cloneencoding human TIE ligand-4 (hTL-4 ATCC Accession No. 98095) wasobtained by restriction enzyme digestion, Southern blotting andhybridization of the hTL-4 clone to coding sequences from mouse TL3,followed by subcloning and sequencing the hybridizing fragments. Codingsequences corresponding to the N-terminal and C-terminal amino acids ofhTL4 were used to design PCR primers (shown below), which in turn wereused for PCR amplification of TL4 from human ovary cDNA. A PCR band wasidentified as corresponding to human TL4 by DNA sequencing using the ABI373A DNA sequencer and Taq Dideoxy Terminator Cycle Sequencing Kit(Applied Biosystems, Inc., Foster City, Calif.). The PCR band was thensubcloned into vector pCR-script and several plasmid clones wereanalyzed by sequencing. The complete human TL4 coding sequence was thencompiled and is shown in FIGS. 23A-23C (SEQ ID NO: 17 and SEQ ID NO:18). In another embodiment of the invention, the nucleotide at position569 is changed from A to G, resulting in an amino acid change from Q toR.

The PCR primers used as described above were designed as follows:hTL4atg 5′-gcatgctatctcgagccaccATGCTCTCCCAGCTAGCCATGCTGCAG-3′(SEQ ID NO:27)

hTL4not5′-gtgtcgacgcggccgctctagatcagacTTAGATGTCCAAAGGCCGTATCATCAT-3′(SEQ ID NO:28)

Lowercase letters indicate “tail” sequences added to the PCR primers tofacilitate cloning of the amplified PCR fragments.

EXAMPLE 23 Construction and Characterization of Modified TIE Ligands

A genetic analysis of TIE-2 ligand-1 and TIE-2 ligand-2 (TL1 and TL2)was undertaken to gain insight into a number of their observedproperties. Although TL1 and TL2 share similar structural homology, theyexhibit different physical and biological properties. The most prominentfeature that distinguishes the two ligands is that although they bothbind to the TIE-2 receptor, TL1 is an agonist while TL2 is anantagonist. Under non-reducing electrophoretic conditions both proteinsexhibit covalent, multimeric structures. TL1 is produced as a mixture ofdisulfide cross-linked multimers, primarily trimers and higher orderspecies, without any dimeric species. But TL2 is produced almostexclusively as a dimeric species. Also, while TL2 is produced well inmost expression systems, TL1 is expressed poorly and is difficult toproduce in large quantities. Finally, production and purificationconditions also appear to predispose TL1 to inactivation by proteolyticcleavage at a site near the amino terminus.

To study these differences, several modified ligands were constructed asfollows.

23.1. Cysteine substitution—Investigations into what factors might becontributing to the different physical and biological properties of thetwo molecules revealed the presence in TL1 of a cysteine residue (CYS265 in FIGS. 4A-4D [SEQ ID NO: 1 and SEQ ID NO: 2]; CYS 245 in FIG. 17[SEQ ID NO: 7 and SEQ ID NO: 8]) preceding the fibrinogen-like domain inTL1 but absent in TL2—i.e., there was no corresponding cysteine residuein TL2. The CYS265 residue in TL1 is encoded by TGC and is located atabout nucleotides 1102-1104 (see FIGS. 4A-4D [SEQ ID NO: 1 and SEQ IDNO: 2]) at the approximate junction between the coiled-coil andfibrinogen-like domains. Because cysteine residues are generallyinvolved in disulfide bond formation, the presence of which cancontribute to both the tertiary structure and biological properties of amolecule, it was thought that perhaps the presence of the CYS265 residuein TL1 might be at least partially responsible for the differentproperties of the two molecules.

To test this hypothesis, an expression plasmid was constructed whichcontained a mutation in TL1 in which the CYS (residue 265 in FIGS. 4A-4D[SEQ ID NO: 1 and SEQ ID NO: 2]; residue 245 in FIG. 17) was replacedwith an amino acid (serine) which does not form disulfide bonds. Inaddition to this TL1/CYS mutant, a second expression plasmid wasconstructed which mutated the approximately corresponding position inTL2 (Met247 in FIG. 17 [SEQ ID NO: 7 and SEQ ID NO: 8]) so that thisresidue was now a cysteine. Both non-mutated and mutated expressionplasmids of TL1 and TL2 were transiently transfected into COS7 cells,cell supernatants containing the recombinant proteins were harvested,and samples were subjected to both reducing and non-reducing SDS/PAGEelectrophoresis and subsequent Western blotting.

FIG. 18 shows the Western blots under non-reducing conditions of bothnon-mutated and mutated TL1 and TL2 proteins, revealing that theTL1/CYS⁻ mutant runs as a dimer much like TL2 and that theTL2/CYS+mutant is able to form a trimer, as well as higher-ordermultimers, more like TL1. When the two mutant proteins were tested fortheir ability to induce phosphorylation in TIE-2 expressing cells, theTL1/CYS⁻ mutant was able to activate the TIE-2 receptor, whereas theTL2/CYS⁺ mutant was not.

Thus, when the cysteine residue (residue 265 in FIGS. 4A-4D [SEQ ID NO:1 and SEQ ID NO: 2]; residue 245 in [SEQ ID NO: 7 and SEQ ID NO: 8]) ofTL1 was genetically altered to a serine, it was found that the covalentstructure of TL1 became similar to that of TL2, i.e., primarily dimeric.The modified TL1 molecule still behaved as an agonist, thus the trimericand/or higher order multimeric structure was not the determining factorgiving TL1 the ability to activate. Although the removal of the cysteinedid make a molecule with more desirable properties, it did not improvethe production level of TH1.

23.2. Domain deletions—The nucleotide sequences encoding TL1 and TL2share a genetic structure that can be divided into three domains, basedon the amino acid sequences of the mature proteins. The lastapproximately 215 amino acid residues of each mature protein containssix cysteines and bears strong resemblance to a domain of fibrinogen.This region was thus denoted the “fibrinogen-like” domain or “F-domain.”A central region of the mature protein containing approximately 205residues had a high probability of assuming a “coiled-coil” structureand was denoted the “coiled-coil” domain or “C-domain.” Theamino-terminal approximately 55 residues of the mature protein containedtwo cysteines and had a low probability of having a coiled-coilstructure. This region was designated the “N-terminal” domain or“N-domain.” The modified ligands described herein are designated using aterminology wherein N=N-terminal domain, C=coiled-coil domain,F=fibrinogen-like domain and the numbers 1 and 2 refer to TL1 and TL2respectively. Thus 1N indicates the N-terminal domain from TL1, 2Findicates the fibrinogen-like domain of TL2, and so forth.

In order to test whether the fibrinogen-like domain (F-domain) of theTIE2 ligands contained TIE-2 activating activity, expression plasmidswere constructed which deleted the coiled-coil and N-terminal domains,leaving only that portion of the DNA sequence encoding the F-domain (forTL1, beginning in FIGS. 4A-4D [SEQ ID NO: 1 and SEQ ID NO: 2] at aboutnucleotide 1159, amino acid residue ARG284; for TL2, corresponding toabout nucleotide 1200 in FIGS. 6A-6D [SEQ ID NO: 5 and SEQ ID NO: 6],amino acid residue 282). This mutant construct was then transientlytransfected into COS cells. The supernatant containing the recombinantprotein was harvested. The TL1/F-domain mutant was tested for itsability to bind the TIE-2 receptor. The results showed that, as amonomer, the TL1/F-domain mutant was not able to bind TIE-2 at adetectable level.

But when the TL1/F-domain monomer was myc-tagged and subsequentlyclustered with an antibody directed against the myc tag, it exhibiteddetectable binding to TIE-2. However, the antibody-clusteredTL1/F-domain mutant was not able to induce phosphorylation in a TIE-2expressing cell line.

Thus it was determined that the F-domain of the TIE-2 ligands isinvolved in binding the receptor but that a truncation consisting ofjust the F-domain alone is not sufficient for receptor binding. Thisraised the possibility that the coiled-coil domain was responsible forholding together several fibrinogen-like domains, which might beessential for receptor binding. In an attempt to confirm thishypothesis, the F-domain was fused with the Fc section of human antibodyIgG1. Because Fc sections dimerize upon expression by mammalian cells,these recombinant proteins mimicked the theoretical configuration of theF-domains were the native ligands to dimerize. This F-domain-Fcconstruct bound but failed to activate the receptor. Apparently,multimerization caused by other regions of the ligands is necessary toenable the ligands to bind the TIE receptor. In addition, some otherfactor outside of the F-domain must contribute to phosphorylation of thereceptor.

Mutants were then constructed which were missing the fibrinogen-likedomain, and therefore contained only the N-terminal and coiled-coildomains. They were not capable of binding to the receptor. To assess therole of the N-terminal domain in receptor binding and activation, theligands were truncated to just their C- and F-domains and tagged with aFLAG tag at the N-terminus, creating constructs termed FLAG-1C1F andFLAG-2C2F. Although these molecules stained robustly in COS7 cellstransfected transiently to express the TIE receptor, they failed torespond in a phosphorylation assay. Thus the N-domain does contain anessential factor for receptor activation although, as disclosed infra,the ability of chimeric molecule 2N2C1F to activate the receptor showsthat even the N-domain of an inactive ligand can fill that role.

The differences in behavior between the myc-tagged F-domain truncationand the Fc-tagged F-domain truncation described previously suggestedthat the TIE ligands can only bind in dimeric or higher multimericforms. Indeed, non-reducing SDS-PAGE showed that the TIE ligands existnaturally in dimeric, trimeric, and multimeric forms. That the FLAG-1C1Fand FLAG-2C2F truncations can bind to the TIE-2 receptor withoutdimerization by a synthetic tag (such as Fc), whereas the F truncationscannot, suggests that the C-region is at least partly responsible forthe aggregation of the F-domains.

23.3. Swapping Constructs (chimeras)

Applicants had noted that the level of production of TL1 in COS7 cellswas approximately tenfold lower than production of TL2. Therefore,chimeras of TL1 and TL2 were constructed in an attempt to explain thisdifference and also to further characterize the agonist activity of TL1as compared to the antagonist activity of TL2.

Four chimeras were constructed in which either the N-terminal domain orthe fibrinogen domain was exchanged between TL1 and TL2 and weredesignated using the terminology described previously such that, forexample, 1N1C2F refers to a chimera having the N-terminal andcoiled-coil domains of TL1, together with the fibrinogen-like domainfrom TL2.

The four chimeras were constructed as follows:

chimera 1—1N1C2F

chimera 2—2N2C1F

chimera 3—1N2C2F

chimera 4—2N1C1F

The nucleotide and amino acid sequences of chimeras 1-4 are shown inFIGS. 24A-24C (SEQ ID NO: 19 and SEQ ID NO: 20), FIGS. 25A-25C (SEQ IDNO: 21 and SEQ ID NO: 22), FIGS. 26A-26C (SEQ ID NO: 23 and SEQ IDNO:24), and FIGS. 27A-27C (SEQ ID NO: 25 and SEQ ID NO: 26)respectively.

Each chimera was inserted into a separate expression vector pJFE14. Thechimeras were then transfected into COS7 cells, along with the emptypJFE14 vector, native TL1, and native TL2 as controls, and the culturesupernatants were collected.

In order to determine how the swapping affected the level of expressionof the ligands, a 1:5 dilution and a 1:50 dilution of the COS7supernatants were dot-blotted onto nitrocellulose. Three ligands thatcontained the TL1 N-domain (i.e. native TL1, 1N2C2F and 1N1C2F) werethen probed with a rabbit antibody specific to the N-terminus of TL1.Three ligands containing the TL2 N-domain, (i.e. native TL2, 2N1C1F and2N2C1F) were probed with a rabbit antibody specific for the N-terminusof TL2. The results demonstrated that the COS7 cells were expressing anymolecule containing the N-domain of TL2 at roughly ten times the levelof any molecule containing the TL1 N-domain, regardless of the makeup ofthe rest of the protein. The conclusion was that the N-domain mustprincipally control the level of expression of the ligand.

The next question addressed was the chimeras' ability or inability toactivate the TIE-2 receptor. EAhy926 cells were challenged with the fourchimeras, as well as TL1 as a positive control for phosphorylation andTL2 or an empty pJFE14-transfected COS7 cell supernatant as negativecontrols for phosphorylation. The cells were lysed, and the TIE-2receptor was immunoprecipitated out of the cell lysate and run on anSDS-PAGE. The samples were Western blotted and probed with ananti-phosphotyrosine antibody to detect any receptors that had beenphosphorylated. Surprisingly, only the constructs containing the TL1fibrinogen-like domain (2N1C1F and 2N2C1F) could phosphorylate the TIE-2receptor. Thus, although the N-terminal region of TL1 is essential foractivation, it can be replaced by the N-terminal region of TL2, i.e.,the information that determines whether the ligand is an agonist or anantagonist is actually contained in the fibrinogen-like domain. Thus itwas determined that the F-domain, in addition to binding the TIE-2receptor, is responsible for the phosphorylation activity of TL1.Further, when TL2, an otherwise inactive molecule, was altered byreplacing its F-domain with the TL1 F-domain, the altered TL2 acted asan agonist.

The 2N1C1F construct was somewhat more potent, however. The signalcaused by chimera 2N1C1F appeared slightly stronger than that of chimera2N2C1F, leading to speculation that the C-domain of TL1, though notcrucial for phosphorylation, might enhance the potency of TL1. However,since the samples used for the phosphorylation assay were not normalizedin terms of the concentration of ligand, it was possible that a strongerphosphorylation signal only indicated the presence of more ligand. Thephosphorylation assay was therefore repeated with varying amounts ofligand to determine whether the active chimeras displayed differentpotencies. The concentration of ligand in the COS7 supernatants ofligand transfections was determined through BIAcore biosenser technologyaccording to methods previously described (Stitt, T. N., et al. (1995)Cell 80: 661-670). BIAcore measured the binding activity of asupernatant to the TIE-2 receptor in arbitrary units called resonanceunits (RU). Fairly good correlation between RU's and ligandconcentration has been generally observed, with 400 RU of activitycorresponding to about 1 μg of protein per mL of supernatant. Sampleswere diluted to concentrations of 100 RU, 20 RU, and 5 RU each and thephosphorylation assay was repeated. The results demonstrated thatchimera 2N2C1F was clearly more potent than either the native TL1 orchimera 1N1C2F at the same concentrations.

Another interesting aspect of these exchange constructs is in theirlevels of expression. Each of the four chimeras was tested for its levelof production in COS cells, its ability to bind to TIE2, and its abilityto phosphorylate TIE2. The results of these experiments showed thatchimeras 1 and 3 were produced at levels comparable to TL1, whereaschimeras 2 and 4 were produced at levels comparable to TL2. Thus a highlevel of protein production was correlated with the TL2 N-terminaldomain. Additionally, when tested on endothelial EAhy926 cells, chimeras2 and 4 were active, whereas 1 and 3 were not. Thus activity(phosphorylation of the receptor) correlates with the TL1fibrinogen-like domain. Chimeras 2 and 4 therefore each had thedesirable properties of high production levels as well as agonistactivity.

23.4. Proteolytic resistant constructs—Based on the observation that alarge fraction of TL1 preparations was often proteolytically cleavednear the N-terminus, it was proposed that an arginine residue located atposition 49 of the mature protein (see FIG. 17 [SEQ ID NO: 7 and SEQ IDNO: 8]) was a candidate cleavage site that might be involved in theregulation of the protein's activity in vixo, and that replacing thearginine with a serine (R49—>S) might increase the stability of theprotein without necessarily affecting its activity. Such a mutant of TL1was constructed and was found to be about as active as the native TL1but did not exhibit resistance to proteolytic cleavage.

23.5. Combination mutants—The most potent of the chimeric constructs,2N1C1F, was additionally altered so that the cysteine encoded bynucleotides 784-786 as shown in FIGS. 27A-27C (SEQ ID NO: 25 and SEQ IDNO: 26) was converted to a serine. This molecule (denoted 2N1C1F(C246S)) was expressed well, potently activated the receptor, wasresistant to proteolytic cleavage and was primarily dimeric, rather thanhigher-order multimeric. Thus the 2N domain appeared to confer proteaseresistance on the molecule. Finally, this molecule was further alteredto eliminate the potentially protease sensitive site encoded bynucleotides 199-201 as shown in FIGS. 27A-27C (SEQ ID NO: 25 and SEQ IDNO: 26), to give a molecule (denoted 2N1C1F (R51->S,C246->S)) which wasexpected to be activating, well expressed, dimeric, and proteaseresistant.

Table 1 summarizes the modified TIE-2 ligand constructs that were madeand characterizes each of them in terms of ability to bind the TIE-2receptor, ability to activate the TIE-2 receptor, the type of structureformed (monomer, dimer, etc.) and their relative production levels.Unmodified TL1 (plain) and TL2 (striped) are shown with the threedomains as boxes. Thus striped boxes indicate domains from TL2. Thecysteine located at position 245 of the mature TL1 protein is indicatedby a “C.” An “X” through the “C” indicates that that cysteine residuewas substituted for by another amino acid as in, for example, the TL1CYS-mutant. Similarly, an “X” through the “R” in the last constructindicates the substitution for an Arg residue at position 49 of themature TL1 protein. The “C.” is present in one modified TL2 constructshowing the TL2 CYS⁺ mutant. Constructs having Fc tails or flag taggingare also indicated.

Based upon the teachings herein, one of skill in the art can readily seethat further constructs may be made in order to create additionalmodified and chimeric TIE-2 ligands which have altered properties. Forexample, one may create a construct comprised of the N-terminal domainof TL2 and the F-domain of TL1 fused with the Fc section of humanantibody IgG1. This construct would be expected to bind and activate theTIE-2 receptor. Similarly, other constructs may be created using theteachings herein and are therefore considered to be within the scope ofthis invention.

23.6. Materials and Methods

Construction of Chimeras

Swapping constructs were inserted into a pJFE14 vector in which the Xbalsite was changed to an AscI site. This vector was then digested withAscI and NotI yielding an AscI-NotI backbone. DNA fragments for thechimeras were generated by PCR using appropriate oligonucleotides.

The FLAG-1C1F and FLAG-2C2F inserts were subcloned into a pMT21 vectorbackbone that had been digested with EcoRI and NotI. The “CF”truncations were obtained through PCR, and the FLAG tag and a precedingtrypsin signalling sequence were constructed by annealing syntheticoligonucleotides.

Transfections

All constructs were transfected transiently into COS7 cells using eitherDEAE-Dextran or LipofectAMINE according to standard protocols. Cellcultures were harvested 3 days after the transfection and spun down at1000 rpm for 1 minute, and the supernatants were transferred to freshtubes and stored at −20° C.

Staining of FLAG-1C1F-Transfected and FLAG-2C2F-Transfected Cells 6-welldishes of COS7 cells were transfected transiently with the TIE-2receptor. The COS7 supernatant from various ligand tansfections wasincubated on the cells for 30 minutes, followed by two washes withPhosphate Buffered Saline (PBS) without magnesium or calcium. The cellswere fixed in −20° C. methanol for 3 minutes, washed once with PBS, andincubated with anti-FLAG M2 antibody (IBI;1:3000 dilution) in PBS/10%Bovine Calf Serum (BCS) for 30 minutes. The cells were washed once withPBS and incubated with goat anti-mouse IgG Alkaline Phosphatase (AP)conjugated antibody (Promega;1:1000) in PBS/10% BCS. The cells werewashed twice with PBS and incubated with the phosphate substrate,BCIP/NBT, with 1 mM levamisole.

Phosphorylation Assays

Dilution of COS7 supernatants for the dose response study was done inthe supernatants of COS7 cells transfected with the empty vector pJFE14.EA cells that naturally express the TIE-2 receptor were starved for >2hours in serum-free medium, followed by challenge with the appropriateCOS7 supernatant for 10 minutes at 37° C. in an atmosphere of 5% CO2.The cells were then rinsed in ice-cold PBS and lysed with 1% NP40 lysisbuffer containing protease inhibitors (10 μg/ml leupeptin, 10 μg/mlaprotinin, 1 mM PMSF) followed by immunoprecipitation with an antibodyspecific for the TIE-2 receptor. Samples were then subjected toimmunoblot analysis, using anti pTyr antibodies.

Dot Blots

Samples were applied to a nitrocellulose membrane, which was blocked andprobed with the appropriate antibodies.

TABLE 1 MUTATION ANALYSIS OF TIE LIGANDS

COILED- FIBRINOGEN- TIE2 TIE2 Multimeric Production N COIL LIKE BindingActivation Structure Levels TL1

+ + HIGHER ORDER LOW TL2

+ − DIMER HIGH

+ + DIMER LOW

+ − HIGHER ORDER HIGH

− N.D. N.D. LOW

− N.D. N.D. HIGH

− − MONOMER HIGH

− − MONOMER HIGH

+ − DIMER HIGH

+ − DIMER HIGH

+ + HIGHER ORDER LOW

+ − HIGHER ORDER LOW flag-

+ + N.D. LOW flag-

+ − N.D. HIGH

+ − N.D. HIGH

+ − N.D. HIGH

+ − N.D. LOW

+ + N.D. HIGH*

+ − N.D. LOW

+ +** N.D. HIGH

+ +** DIMER HIGH

+ + N.D. LOW *HIGHEST PRODUCTION OF RU **MOST POTENTLY ACTIVATING N.D. =NOT DETERMINED

Deposits

The following have been deposited with the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md. 20852 in accordancewith the Budapest Treaty. A plasmid clone encoding a TIE-2 ligand wasdeposited with the ATCC on Oct. 7, 1994 and designated as “pJFE14encoding TIE-2 ligand” under ATCC Accession No. 75910. RecombinantAutographa californica baculovirus encoding TIE-2 receptorbody wasdeposited with the ATCC on Oct. 7, 1994 and designated as “vTIE-2receptorbody” under ATCC Accession No. VR2484. A lambda phage vectorcontaining human tie-2 ligand DNA was deposited with the ATCC on Oct.26, 1994 and designated as “λgt10 encoding htie-2 ligand 1” under ATCCAccession No. 75928. A plasmid clone encoding a second TIE-2 ligand wasdeposited with the ATCC on Dec. 9, 1994 and designated as “pBluescriptKS encoding human TIE 2 ligand 2” under ATCC Accession No. 75963. E.coli strain DH10B containing plasmid pBeLoBac11 with a human TL-4 geneinsert encoding human TIE ligand-4 was deposited with the ATCC on Jul.2, 1996 and designated as “hTL-4” under ATCC Accession No. 98095.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

30 1 2149 DNA Homo sapiens CDS (310)..(1803) 1 cagctgactc aggcaggctccatgctgaac ggtcacacag agaggaaaca ataaatctca 60 gctactatgc aataaatatctcaagtttta acgaagaaaa acatcattgc agtgaaataa 120 aaaattttaa aattttagaacaaagctaac aaatggctag ttttctatga ttcttcttca 180 aacgctttct ttgagggggaaagagtcaaa caaacaagca gttttacctg aaataaagaa 240 ctagttttag aggtcagaagaaaggagcaa gttttgcgag aggcacggaa ggagtgtgct 300 ggcagtaca atg aca gttttc ctt tcc ttt gct ttc ctc gct gcc att ctg 351 Met Thr Val Phe Leu SerPhe Ala Phe Leu Ala Ala Ile Leu 1 5 10 act cac ata ggg tgc agc aat cagcgc cga agt cca gaa aac agt ggg 399 Thr His Ile Gly Cys Ser Asn Gln ArgArg Ser Pro Glu Asn Ser Gly 15 20 25 30 aga aga tat aac cgg att caa catggg caa tgt gcc tac act ttc att 447 Arg Arg Tyr Asn Arg Ile Gln His GlyGln Cys Ala Tyr Thr Phe Ile 35 40 45 ctt cca gaa cac gat ggc aac tgt cgtgag agt acg aca gac cag tac 495 Leu Pro Glu His Asp Gly Asn Cys Arg GluSer Thr Thr Asp Gln Tyr 50 55 60 aac aca aac gct ctg cag aga gat gct ccacac gtg gaa ccg gat ttc 543 Asn Thr Asn Ala Leu Gln Arg Asp Ala Pro HisVal Glu Pro Asp Phe 65 70 75 tct tcc cag aaa ctt caa cat ctg gaa cat gtgatg gaa aat tat act 591 Ser Ser Gln Lys Leu Gln His Leu Glu His Val MetGlu Asn Tyr Thr 80 85 90 cag tgg ctg caa aaa ctt gag aat tac att gtg gaaaac atg aag tcg 639 Gln Trp Leu Gln Lys Leu Glu Asn Tyr Ile Val Glu AsnMet Lys Ser 95 100 105 110 gag atg gcc cag ata cag cag aat gca gtt cagaac cac acg gct acc 687 Glu Met Ala Gln Ile Gln Gln Asn Ala Val Gln AsnHis Thr Ala Thr 115 120 125 atg ctg gag ata gga acc agc ctc ctc tct cagact gca gag cag acc 735 Met Leu Glu Ile Gly Thr Ser Leu Leu Ser Gln ThrAla Glu Gln Thr 130 135 140 aga aag ctg aca gat gtt gag acc cag gta ctaaat caa act tct cga 783 Arg Lys Leu Thr Asp Val Glu Thr Gln Val Leu AsnGln Thr Ser Arg 145 150 155 ctt gag ata cag ctg ctg gag aat tca tta tccacc tac aag cta gag 831 Leu Glu Ile Gln Leu Leu Glu Asn Ser Leu Ser ThrTyr Lys Leu Glu 160 165 170 aag caa ctt ctt caa cag aca aat gaa atc ttgaag atc cat gaa aaa 879 Lys Gln Leu Leu Gln Gln Thr Asn Glu Ile Leu LysIle His Glu Lys 175 180 185 190 aac agt tta tta gaa cat aaa atc tta gaaatg gaa gga aaa cac aag 927 Asn Ser Leu Leu Glu His Lys Ile Leu Glu MetGlu Gly Lys His Lys 195 200 205 gaa gag ttg gac acc tta aag gaa gag aaagag aac ctt caa ggc ttg 975 Glu Glu Leu Asp Thr Leu Lys Glu Glu Lys GluAsn Leu Gln Gly Leu 210 215 220 gtt act cgt caa aca tat ata atc cag gagctg gaa aag caa tta aac 1023 Val Thr Arg Gln Thr Tyr Ile Ile Gln Glu LeuGlu Lys Gln Leu Asn 225 230 235 aga gct acc acc aac aac agt gtc ctt cagaag cag caa ctg gag ctg 1071 Arg Ala Thr Thr Asn Asn Ser Val Leu Gln LysGln Gln Leu Glu Leu 240 245 250 atg gac aca gtc cac aac ctt gtc aat ctttgc act aaa gaa ggt gtt 1119 Met Asp Thr Val His Asn Leu Val Asn Leu CysThr Lys Glu Gly Val 255 260 265 270 tta cta aag gga gga aaa aga gag gaagag aaa cca ttt aga gac tgt 1167 Leu Leu Lys Gly Gly Lys Arg Glu Glu GluLys Pro Phe Arg Asp Cys 275 280 285 gca gat gta tat caa gct ggt ttt aataaa agt gga atc tac act att 1215 Ala Asp Val Tyr Gln Ala Gly Phe Asn LysSer Gly Ile Tyr Thr Ile 290 295 300 tat att aat aat atg cca gaa ccc aaaaag gtg ttt tgc aat atg gat 1263 Tyr Ile Asn Asn Met Pro Glu Pro Lys LysVal Phe Cys Asn Met Asp 305 310 315 gtc aat ggg gga ggt tgg act gta atacaa cat cgt gaa gat gga agt 1311 Val Asn Gly Gly Gly Trp Thr Val Ile GlnHis Arg Glu Asp Gly Ser 320 325 330 cta gat ttc caa aga ggc tgg aag gaatat aaa atg ggt ttt gga aat 1359 Leu Asp Phe Gln Arg Gly Trp Lys Glu TyrLys Met Gly Phe Gly Asn 335 340 345 350 ccc tcc ggt gaa tat tgg ctg gggaat gag ttt att ttt gcc att acc 1407 Pro Ser Gly Glu Tyr Trp Leu Gly AsnGlu Phe Ile Phe Ala Ile Thr 355 360 365 agt cag agg cag tac atg cta agaatt gag tta atg gac tgg gaa ggg 1455 Ser Gln Arg Gln Tyr Met Leu Arg IleGlu Leu Met Asp Trp Glu Gly 370 375 380 aac cga gcc tat tca cag tat gacaga ttc cac ata gga aat gaa aag 1503 Asn Arg Ala Tyr Ser Gln Tyr Asp ArgPhe His Ile Gly Asn Glu Lys 385 390 395 caa aac tat agg ttg tat tta aaaggt cac act ggg aca gca gga aaa 1551 Gln Asn Tyr Arg Leu Tyr Leu Lys GlyHis Thr Gly Thr Ala Gly Lys 400 405 410 cag agc agc ctg atc tta cac ggtgct gat ttc agc act aaa gat gct 1599 Gln Ser Ser Leu Ile Leu His Gly AlaAsp Phe Ser Thr Lys Asp Ala 415 420 425 430 gat aat gac aac tgt atg tgcaaa tgt gcc ctc atg tta aca gga gga 1647 Asp Asn Asp Asn Cys Met Cys LysCys Ala Leu Met Leu Thr Gly Gly 435 440 445 tgg tgg ttt gat gct tgt ggcccc tcc aat cta aat gga atg ttc tat 1695 Trp Trp Phe Asp Ala Cys Gly ProSer Asn Leu Asn Gly Met Phe Tyr 450 455 460 act gcg gga caa aac cat ggaaaa ctg aat ggg ata aag tgg cac tac 1743 Thr Ala Gly Gln Asn His Gly LysLeu Asn Gly Ile Lys Trp His Tyr 465 470 475 ttc aaa ggg ccc agt tac tcctta cgt tcc aca act atg atg att cga 1791 Phe Lys Gly Pro Ser Tyr Ser LeuArg Ser Thr Thr Met Met Ile Arg 480 485 490 cct tta gat ttt tgaaagcgcaatgtcagaag cgattatgaa agcaacaaag 1843 Pro Leu Asp Phe 495 aaatccggagaagctgccag gtgagaaact gtttgaaaac ttcagaagca aacaatattg 1903 tctcccttccagcaataagt ggtagttatg tgaagtcacc aaggttcttg accgtgaatc 1963 tggagccgtttgagttcaca agagtctcta cttggggtga cagtgctcac gtggctcgac 2023 tatagaaaactccactgact gtcgggcttt aaaaagggaa gaaactgctg agcttgctgt 2083 gcttcaaactactactggac cttattttgg aactatggta gccagatgat aaatatggtt 2143 aatttc 21492 498 PRT Homo sapiens 2 Met Thr Val Phe Leu Ser Phe Ala Phe Leu Ala AlaIle Leu Thr His 1 5 10 15 Ile Gly Cys Ser Asn Gln Arg Arg Ser Pro GluAsn Ser Gly Arg Arg 20 25 30 Tyr Asn Arg Ile Gln His Gly Gln Cys Ala TyrThr Phe Ile Leu Pro 35 40 45 Glu His Asp Gly Asn Cys Arg Glu Ser Thr ThrAsp Gln Tyr Asn Thr 50 55 60 Asn Ala Leu Gln Arg Asp Ala Pro His Val GluPro Asp Phe Ser Ser 65 70 75 80 Gln Lys Leu Gln His Leu Glu His Val MetGlu Asn Tyr Thr Gln Trp 85 90 95 Leu Gln Lys Leu Glu Asn Tyr Ile Val GluAsn Met Lys Ser Glu Met 100 105 110 Ala Gln Ile Gln Gln Asn Ala Val GlnAsn His Thr Ala Thr Met Leu 115 120 125 Glu Ile Gly Thr Ser Leu Leu SerGln Thr Ala Glu Gln Thr Arg Lys 130 135 140 Leu Thr Asp Val Glu Thr GlnVal Leu Asn Gln Thr Ser Arg Leu Glu 145 150 155 160 Ile Gln Leu Leu GluAsn Ser Leu Ser Thr Tyr Lys Leu Glu Lys Gln 165 170 175 Leu Leu Gln GlnThr Asn Glu Ile Leu Lys Ile His Glu Lys Asn Ser 180 185 190 Leu Leu GluHis Lys Ile Leu Glu Met Glu Gly Lys His Lys Glu Glu 195 200 205 Leu AspThr Leu Lys Glu Glu Lys Glu Asn Leu Gln Gly Leu Val Thr 210 215 220 ArgGln Thr Tyr Ile Ile Gln Glu Leu Glu Lys Gln Leu Asn Arg Ala 225 230 235240 Thr Thr Asn Asn Ser Val Leu Gln Lys Gln Gln Leu Glu Leu Met Asp 245250 255 Thr Val His Asn Leu Val Asn Leu Cys Thr Lys Glu Gly Val Leu Leu260 265 270 Lys Gly Gly Lys Arg Glu Glu Glu Lys Pro Phe Arg Asp Cys AlaAsp 275 280 285 Val Tyr Gln Ala Gly Phe Asn Lys Ser Gly Ile Tyr Thr IleTyr Ile 290 295 300 Asn Asn Met Pro Glu Pro Lys Lys Val Phe Cys Asn MetAsp Val Asn 305 310 315 320 Gly Gly Gly Trp Thr Val Ile Gln His Arg GluAsp Gly Ser Leu Asp 325 330 335 Phe Gln Arg Gly Trp Lys Glu Tyr Lys MetGly Phe Gly Asn Pro Ser 340 345 350 Gly Glu Tyr Trp Leu Gly Asn Glu PheIle Phe Ala Ile Thr Ser Gln 355 360 365 Arg Gln Tyr Met Leu Arg Ile GluLeu Met Asp Trp Glu Gly Asn Arg 370 375 380 Ala Tyr Ser Gln Tyr Asp ArgPhe His Ile Gly Asn Glu Lys Gln Asn 385 390 395 400 Tyr Arg Leu Tyr LeuLys Gly His Thr Gly Thr Ala Gly Lys Gln Ser 405 410 415 Ser Leu Ile LeuHis Gly Ala Asp Phe Ser Thr Lys Asp Ala Asp Asn 420 425 430 Asp Asn CysMet Cys Lys Cys Ala Leu Met Leu Thr Gly Gly Trp Trp 435 440 445 Phe AspAla Cys Gly Pro Ser Asn Leu Asn Gly Met Phe Tyr Thr Ala 450 455 460 GlyGln Asn His Gly Lys Leu Asn Gly Ile Lys Trp His Tyr Phe Lys 465 470 475480 Gly Pro Ser Tyr Ser Leu Arg Ser Thr Thr Met Met Ile Arg Pro Leu 485490 495 Asp Phe 3 2146 DNA Homo sapiens CDS (310)..(1803) 3 cagctgactcaggcaggctc catgctgaac ggtcacacag agaggaaaca ataaatctca 60 gctactatgcaataaatatc tcaagtttta acgaagaaaa acatcattgc agtgaaataa 120 aaaattttaaaattttagaa caaagctaac aaatggctag ttttctatga ttcttcttca 180 aacgctttctttgaggggga aagagtcaaa caaacaagca gttttacctg aaataaagaa 240 ctagttttagaggtcagaag aaaggagcaa gttttgcgag aggcacggaa ggagtgtgct 300 ggcagtaca atgaca gtt ttc ctt tcc ttt gct ttc ctc gct gcc att ctg 351 Met Thr Val PheLeu Ser Phe Ala Phe Leu Ala Ala Ile Leu 1 5 10 act cac ata ggg tgc agcaat cag cgc cga agt cca gaa aac agt ggg 399 Thr His Ile Gly Cys Ser AsnGln Arg Arg Ser Pro Glu Asn Ser Gly 15 20 25 30 aga aga tat aac cgg attcaa cat ggg caa tgt gcc tac act ttc att 447 Arg Arg Tyr Asn Arg Ile GlnHis Gly Gln Cys Ala Tyr Thr Phe Ile 35 40 45 ctt cca gaa cac gat ggc aactgt cgt gag agt acg aca gac cag tac 495 Leu Pro Glu His Asp Gly Asn CysArg Glu Ser Thr Thr Asp Gln Tyr 50 55 60 aac aca aac gct ctg cag aga gatgct cca cac gtg gaa ccg gat ttc 543 Asn Thr Asn Ala Leu Gln Arg Asp AlaPro His Val Glu Pro Asp Phe 65 70 75 tct tcc cag aaa ctt caa cat ctg gaacat gtg atg gaa aat tat act 591 Ser Ser Gln Lys Leu Gln His Leu Glu HisVal Met Glu Asn Tyr Thr 80 85 90 cag tgg ctg caa aaa ctt gag aat tac attgtg gaa aac atg aag tcg 639 Gln Trp Leu Gln Lys Leu Glu Asn Tyr Ile ValGlu Asn Met Lys Ser 95 100 105 110 gag atg gcc cag ata cag cag aat gcagtt cag aac cac acg gct acc 687 Glu Met Ala Gln Ile Gln Gln Asn Ala ValGln Asn His Thr Ala Thr 115 120 125 atg ctg gag ata gga acc agc ctc ctctct cag act gca gag cag acc 735 Met Leu Glu Ile Gly Thr Ser Leu Leu SerGln Thr Ala Glu Gln Thr 130 135 140 aga aag ctg aca gat gtt gag acc caggta cta aat caa act tct cga 783 Arg Lys Leu Thr Asp Val Glu Thr Gln ValLeu Asn Gln Thr Ser Arg 145 150 155 ctt gag ata cag ctg ctg gag aat tcatta tcc acc tac aag cta gag 831 Leu Glu Ile Gln Leu Leu Glu Asn Ser LeuSer Thr Tyr Lys Leu Glu 160 165 170 aag caa ctt ctt caa cag aca aat gaaatc ttg aag atc cat gaa aaa 879 Lys Gln Leu Leu Gln Gln Thr Asn Glu IleLeu Lys Ile His Glu Lys 175 180 185 190 aac agt tta tta gaa cat aaa atctta gaa atg gaa gga aaa cac aag 927 Asn Ser Leu Leu Glu His Lys Ile LeuGlu Met Glu Gly Lys His Lys 195 200 205 gaa gag ttg gac acc tta aag gaagag aaa gag aac ctt caa ggc ttg 975 Glu Glu Leu Asp Thr Leu Lys Glu GluLys Glu Asn Leu Gln Gly Leu 210 215 220 gtt act cgt caa aca tat ata atccag gag ctg gaa aag caa tta aac 1023 Val Thr Arg Gln Thr Tyr Ile Ile GlnGlu Leu Glu Lys Gln Leu Asn 225 230 235 aga gct acc acc aac aac agt gtcctt cag aag cag caa ctg gag ctg 1071 Arg Ala Thr Thr Asn Asn Ser Val LeuGln Lys Gln Gln Leu Glu Leu 240 245 250 atg gac aca gtc cac aac ctt gtcaat ctt tgc act aaa gaa gtt tta 1119 Met Asp Thr Val His Asn Leu Val AsnLeu Cys Thr Lys Glu Val Leu 255 260 265 270 cta aag gga gga aaa aga gaggaa gag aaa cca ttt aga gac tgt gca 1167 Leu Lys Gly Gly Lys Arg Glu GluGlu Lys Pro Phe Arg Asp Cys Ala 275 280 285 gat gta tat caa gct ggt tttaat aaa agt gga atc tac act att tat 1215 Asp Val Tyr Gln Ala Gly Phe AsnLys Ser Gly Ile Tyr Thr Ile Tyr 290 295 300 att aat aat atg cca gaa cccaaa aag gtg ttt tgc aat atg gat gtc 1263 Ile Asn Asn Met Pro Glu Pro LysLys Val Phe Cys Asn Met Asp Val 305 310 315 aat ggg gga ggt tgg act gtaata caa cat cgt gaa gat gga agt cta 1311 Asn Gly Gly Gly Trp Thr Val IleGln His Arg Glu Asp Gly Ser Leu 320 325 330 gat ttc caa aga ggc tgg aaggaa tat aaa atg ggt ttt gga aat ccc 1359 Asp Phe Gln Arg Gly Trp Lys GluTyr Lys Met Gly Phe Gly Asn Pro 335 340 345 350 tcc ggt gaa tat tgg ctgggg aat gag ttt att ttt gcc att acc agt 1407 Ser Gly Glu Tyr Trp Leu GlyAsn Glu Phe Ile Phe Ala Ile Thr Ser 355 360 365 cag agg cag tac atg ctaaga att gag tta atg gac tgg gaa ggg aac 1455 Gln Arg Gln Tyr Met Leu ArgIle Glu Leu Met Asp Trp Glu Gly Asn 370 375 380 cga gcc tat tca cag tatgac aga ttc cac ata gga aat gaa aag caa 1503 Arg Ala Tyr Ser Gln Tyr AspArg Phe His Ile Gly Asn Glu Lys Gln 385 390 395 aac tat agg ttg tat ttaaaa ggt cac act ggg aca gca gga aaa cag 1551 Asn Tyr Arg Leu Tyr Leu LysGly His Thr Gly Thr Ala Gly Lys Gln 400 405 410 agc agc ctg atc tta cacggt gct gat ttc agc act aaa gat gct gat 1599 Ser Ser Leu Ile Leu His GlyAla Asp Phe Ser Thr Lys Asp Ala Asp 415 420 425 430 aat gac aac tgt atgtgc aaa tgt gcc ctc atg tta aca gga gga tgg 1647 Asn Asp Asn Cys Met CysLys Cys Ala Leu Met Leu Thr Gly Gly Trp 435 440 445 tgg ttt gat gct tgtggc ccc tcc aat cta aat gga atg ttc tat act 1695 Trp Phe Asp Ala Cys GlyPro Ser Asn Leu Asn Gly Met Phe Tyr Thr 450 455 460 gcg gga caa aac catcga aaa ctg aat ggg ata aag tgg cac tac ttc 1743 Ala Gly Gln Asn His ArgLys Leu Asn Gly Ile Lys Trp His Tyr Phe 465 470 475 aaa ggg ccc agt tactcc tta cgt tcc aca act atg atg att cga cct 1791 Lys Gly Pro Ser Tyr SerLeu Arg Ser Thr Thr Met Met Ile Arg Pro 480 485 490 tta gat ttt tgaaagcgcaatg tcagaagcga ttatgaaagc aacaaagaaa 1843 Leu Asp Phe 495tccggagaag ctgccaggtg agaaactgtt tgaaaacttc agaagcaaac aatattgtct 1903cccttccacc aataagtggt agttatgtga agtcaccaag gttcttgacc gtgaatctgg 1963agccgtttga gttcacaaga gtctctactt ggggtgacag tgctcacgtg gctcgactat 2023agaaaactcc actgactgtc gggctttaaa aagggaagaa actgctgagc ttgctgtgct 2083tcaaactact actggacctt attttggaac tatggtagcc agatgataaa tatggttaat 2143ttc 2146 4 497 PRT Homo sapiens 4 Met Thr Val Phe Leu Ser Phe Ala PheLeu Ala Ala Ile Leu Thr His 1 5 10 15 Ile Gly Cys Ser Asn Gln Arg ArgSer Pro Glu Asn Ser Gly Arg Arg 20 25 30 Tyr Asn Arg Ile Gln His Gly GlnCys Ala Tyr Thr Phe Ile Leu Pro 35 40 45 Glu His Asp Gly Asn Cys Arg GluSer Thr Thr Asp Gln Tyr Asn Thr 50 55 60 Asn Ala Leu Gln Arg Asp Ala ProHis Val Glu Pro Asp Phe Ser Ser 65 70 75 80 Gln Lys Leu Gln His Leu GluHis Val Met Glu Asn Tyr Thr Gln Trp 85 90 95 Leu Gln Lys Leu Glu Asn TyrIle Val Glu Asn Met Lys Ser Glu Met 100 105 110 Ala Gln Ile Gln Gln AsnAla Val Gln Asn His Thr Ala Thr Met Leu 115 120 125 Glu Ile Gly Thr SerLeu Leu Ser Gln Thr Ala Glu Gln Thr Arg Lys 130 135 140 Leu Thr Asp ValGlu Thr Gln Val Leu Asn Gln Thr Ser Arg Leu Glu 145 150 155 160 Ile GlnLeu Leu Glu Asn Ser Leu Ser Thr Tyr Lys Leu Glu Lys Gln 165 170 175 LeuLeu Gln Gln Thr Asn Glu Ile Leu Lys Ile His Glu Lys Asn Ser 180 185 190Leu Leu Glu His Lys Ile Leu Glu Met Glu Gly Lys His Lys Glu Glu 195 200205 Leu Asp Thr Leu Lys Glu Glu Lys Glu Asn Leu Gln Gly Leu Val Thr 210215 220 Arg Gln Thr Tyr Ile Ile Gln Glu Leu Glu Lys Gln Leu Asn Arg Ala225 230 235 240 Thr Thr Asn Asn Ser Val Leu Gln Lys Gln Gln Leu Glu LeuMet Asp 245 250 255 Thr Val His Asn Leu Val Asn Leu Cys Thr Lys Glu ValLeu Leu Lys 260 265 270 Gly Gly Lys Arg Glu Glu Glu Lys Pro Phe Arg AspCys Ala Asp Val 275 280 285 Tyr Gln Ala Gly Phe Asn Lys Ser Gly Ile TyrThr Ile Tyr Ile Asn 290 295 300 Asn Met Pro Glu Pro Lys Lys Val Phe CysAsn Met Asp Val Asn Gly 305 310 315 320 Gly Gly Trp Thr Val Ile Gln HisArg Glu Asp Gly Ser Leu Asp Phe 325 330 335 Gln Arg Gly Trp Lys Glu TyrLys Met Gly Phe Gly Asn Pro Ser Gly 340 345 350 Glu Tyr Trp Leu Gly AsnGlu Phe Ile Phe Ala Ile Thr Ser Gln Arg 355 360 365 Gln Tyr Met Leu ArgIle Glu Leu Met Asp Trp Glu Gly Asn Arg Ala 370 375 380 Tyr Ser Gln TyrAsp Arg Phe His Ile Gly Asn Glu Lys Gln Asn Tyr 385 390 395 400 Arg LeuTyr Leu Lys Gly His Thr Gly Thr Ala Gly Lys Gln Ser Ser 405 410 415 LeuIle Leu His Gly Ala Asp Phe Ser Thr Lys Asp Ala Asp Asn Asp 420 425 430Asn Cys Met Cys Lys Cys Ala Leu Met Leu Thr Gly Gly Trp Trp Phe 435 440445 Asp Ala Cys Gly Pro Ser Asn Leu Asn Gly Met Phe Tyr Thr Ala Gly 450455 460 Gln Asn His Arg Lys Leu Asn Gly Ile Lys Trp His Tyr Phe Lys Gly465 470 475 480 Pro Ser Tyr Ser Leu Arg Ser Thr Thr Met Met Ile Arg ProLeu Asp 485 490 495 Phe 5 2282 DNA Homo sapiens CDS (357)..(1844) 5gaattcctgg gttggtgttt atctcctccc agccttgagg gagggaacaa cactgtagga 60tctggggaga gaggaacaaa ggaccgtgaa agctgctctg taaaagctga cacagccctc 120ccaagtgagc aggactgttc ttcccactgc aatctgacag tttactgcat gcctggagag 180aacacagcag taaaaaccag gtttgctact ggaaaaagag gaaagagaag actttcattg 240acggacccag ccatggcagc gtagcagccc tgcgtttcag acggcagcag ctcgggactc 300tggacgtgtg tttgccctca agtttgctaa gctgctggtt tattactgaa gaaaga atg 359Met 1 tgg cag att gtt ttc ttt act ctg agc tgt gat ctt gtc ttg gcc gca407 Trp Gln Ile Val Phe Phe Thr Leu Ser Cys Asp Leu Val Leu Ala Ala 5 1015 gcc tat aac aac ttt cgg aag agc atg gac agc ata gga aag aag caa 455Ala Tyr Asn Asn Phe Arg Lys Ser Met Asp Ser Ile Gly Lys Lys Gln 20 25 30tat cag gtc cag cat ggg tcc tgc agc tac act ttc ctc ctg cca gag 503 TyrGln Val Gln His Gly Ser Cys Ser Tyr Thr Phe Leu Leu Pro Glu 35 40 45 atggac aac tgc cgc tct tcc tcc agc ccc tac gtg tcc aat gct gtg 551 Met AspAsn Cys Arg Ser Ser Ser Ser Pro Tyr Val Ser Asn Ala Val 50 55 60 65 cagagg gac gcg ccg ctc gaa tac gat gac tcg gtg cag agg ctg caa 599 Gln ArgAsp Ala Pro Leu Glu Tyr Asp Asp Ser Val Gln Arg Leu Gln 70 75 80 gtg ctggag aac atc atg gaa aac aac act cag tgg cta atg aag ctt 647 Val Leu GluAsn Ile Met Glu Asn Asn Thr Gln Trp Leu Met Lys Leu 85 90 95 gag aat tatatc cag gac aac atg aag aaa gaa atg gta gag ata cag 695 Glu Asn Tyr IleGln Asp Asn Met Lys Lys Glu Met Val Glu Ile Gln 100 105 110 cag aat gcagta cag aac cag acg gct gtg atg ata gaa ata ggg aca 743 Gln Asn Ala ValGln Asn Gln Thr Ala Val Met Ile Glu Ile Gly Thr 115 120 125 aac ctg ttgaac caa aca gct gag caa acg cgg aag tta act gat gtg 791 Asn Leu Leu AsnGln Thr Ala Glu Gln Thr Arg Lys Leu Thr Asp Val 130 135 140 145 gaa gcccaa gta tta aat cag acc acg aga ctt gaa ctt cag ctc ttg 839 Glu Ala GlnVal Leu Asn Gln Thr Thr Arg Leu Glu Leu Gln Leu Leu 150 155 160 gaa cactcc ctc tcg aca aac aaa ttg gaa aaa cag att ttg gac cag 887 Glu His SerLeu Ser Thr Asn Lys Leu Glu Lys Gln Ile Leu Asp Gln 165 170 175 acc agtgaa ata aac aaa ttg caa gat aag aac agt ttc cta gaa aag 935 Thr Ser GluIle Asn Lys Leu Gln Asp Lys Asn Ser Phe Leu Glu Lys 180 185 190 aag gtgcta gct atg gaa gac aag cac atc atc caa cta cag tca ata 983 Lys Val LeuAla Met Glu Asp Lys His Ile Ile Gln Leu Gln Ser Ile 195 200 205 aaa gaagag aaa gat cag cta cag gtg tta gta tcc aag caa aat tcc 1031 Lys Glu GluLys Asp Gln Leu Gln Val Leu Val Ser Lys Gln Asn Ser 210 215 220 225 atcatt gaa gaa cta gaa aaa aaa ata gtg act gcc acg gtg aat aat 1079 Ile IleGlu Glu Leu Glu Lys Lys Ile Val Thr Ala Thr Val Asn Asn 230 235 240 tcagtt ctt caa aag cag caa cat gat ctc atg gag aca gtt aat aac 1127 Ser ValLeu Gln Lys Gln Gln His Asp Leu Met Glu Thr Val Asn Asn 245 250 255 ttactg act atg atg tcc aca tca aac tca gct aag gac ccc act gtt 1175 Leu LeuThr Met Met Ser Thr Ser Asn Ser Ala Lys Asp Pro Thr Val 260 265 270 gctaaa gaa gaa caa atc agc ttc aga gac tgt gct gaa gta ttc aaa 1223 Ala LysGlu Glu Gln Ile Ser Phe Arg Asp Cys Ala Glu Val Phe Lys 275 280 285 tcagga cac acc aca aat ggc atc tac acg tta aca ttc cct aat tct 1271 Ser GlyHis Thr Thr Asn Gly Ile Tyr Thr Leu Thr Phe Pro Asn Ser 290 295 300 305aca gaa gag atc aag gcc tac tgt gac atg gaa gct gga gga ggc ggg 1319 ThrGlu Glu Ile Lys Ala Tyr Cys Asp Met Glu Ala Gly Gly Gly Gly 310 315 320tgg aca att att cag cga cgt gag gat ggc agc gtt gat ttt cag agg 1367 TrpThr Ile Ile Gln Arg Arg Glu Asp Gly Ser Val Asp Phe Gln Arg 325 330 335act tgg aaa gaa tat aaa gtg gga ttt ggt aac cct tca gga gaa tat 1415 ThrTrp Lys Glu Tyr Lys Val Gly Phe Gly Asn Pro Ser Gly Glu Tyr 340 345 350tgg ctg gga aat gag ttt gtt tcg caa ctg act aat cag caa cgc tat 1463 TrpLeu Gly Asn Glu Phe Val Ser Gln Leu Thr Asn Gln Gln Arg Tyr 355 360 365gtg ctt aaa ata cac ctt aaa gac tgg gaa ggg aat gag gct tac tca 1511 ValLeu Lys Ile His Leu Lys Asp Trp Glu Gly Asn Glu Ala Tyr Ser 370 375 380385 ttg tat gaa cat ttc tat ctc tca agt gaa gaa ctc aat tat agg att 1559Leu Tyr Glu His Phe Tyr Leu Ser Ser Glu Glu Leu Asn Tyr Arg Ile 390 395400 cac ctt aaa gga ctt aca ggg aca gcc ggc aaa ata agc agc atc agc 1607His Leu Lys Gly Leu Thr Gly Thr Ala Gly Lys Ile Ser Ser Ile Ser 405 410415 caa cca gga aat gat ttt agc aca aag gat gga gac aac gac aaa tgt 1655Gln Pro Gly Asn Asp Phe Ser Thr Lys Asp Gly Asp Asn Asp Lys Cys 420 425430 att tgc aaa tgt tca caa atg cta aca gga ggc tgg tgg ttt gat gca 1703Ile Cys Lys Cys Ser Gln Met Leu Thr Gly Gly Trp Trp Phe Asp Ala 435 440445 tgt ggt cct tcc aac ttg aac gga atg tac tat cca cag agg cag aac 1751Cys Gly Pro Ser Asn Leu Asn Gly Met Tyr Tyr Pro Gln Arg Gln Asn 450 455460 465 aca aat aag ttc aac ggc att aaa tgg tac tac tgg aaa ggc tca ggc1799 Thr Asn Lys Phe Asn Gly Ile Lys Trp Tyr Tyr Trp Lys Gly Ser Gly 470475 480 tat tcg ctc aag gcc aca acc atg atg atc cga cca gca gat ttc 1844Tyr Ser Leu Lys Ala Thr Thr Met Met Ile Arg Pro Ala Asp Phe 485 490 495taaacatccc agtccacctg aggaactgtc tcgaactatt ttcaaagact taagcccagt 1904gcactgaaag tcacggctgc gcactgtgtc ctcttccacc acagagggcg tgtgctcggt 1964gctgacggga cccacatgct ccagattaga gcctgtaaac tttatcactt aaacttgcat 2024cacttaacgg accaaagcaa gaccctaaac atccataatt gtgattagac agaacaccta 2084tgcaaagatg aacccgaggc tgagaatcag actgacagtt tacagacgct gctgtcacaa 2144ccaagaatgt tatgtgcaag tttatcagta aataactgga aaacagaaca cttatgttat 2204acaatacaga tcatcttgga actgcattct tctgagcact gtttatacac tgtgtaaata 2264cccatatgtc ctgaattc 2282 6 496 PRT Homo sapiens 6 Met Trp Gln Ile ValPhe Phe Thr Leu Ser Cys Asp Leu Val Leu Ala 1 5 10 15 Ala Ala Tyr AsnAsn Phe Arg Lys Ser Met Asp Ser Ile Gly Lys Lys 20 25 30 Gln Tyr Gln ValGln His Gly Ser Cys Ser Tyr Thr Phe Leu Leu Pro 35 40 45 Glu Met Asp AsnCys Arg Ser Ser Ser Ser Pro Tyr Val Ser Asn Ala 50 55 60 Val Gln Arg AspAla Pro Leu Glu Tyr Asp Asp Ser Val Gln Arg Leu 65 70 75 80 Gln Val LeuGlu Asn Ile Met Glu Asn Asn Thr Gln Trp Leu Met Lys 85 90 95 Leu Glu AsnTyr Ile Gln Asp Asn Met Lys Lys Glu Met Val Glu Ile 100 105 110 Gln GlnAsn Ala Val Gln Asn Gln Thr Ala Val Met Ile Glu Ile Gly 115 120 125 ThrAsn Leu Leu Asn Gln Thr Ala Glu Gln Thr Arg Lys Leu Thr Asp 130 135 140Val Glu Ala Gln Val Leu Asn Gln Thr Thr Arg Leu Glu Leu Gln Leu 145 150155 160 Leu Glu His Ser Leu Ser Thr Asn Lys Leu Glu Lys Gln Ile Leu Asp165 170 175 Gln Thr Ser Glu Ile Asn Lys Leu Gln Asp Lys Asn Ser Phe LeuGlu 180 185 190 Lys Lys Val Leu Ala Met Glu Asp Lys His Ile Ile Gln LeuGln Ser 195 200 205 Ile Lys Glu Glu Lys Asp Gln Leu Gln Val Leu Val SerLys Gln Asn 210 215 220 Ser Ile Ile Glu Glu Leu Glu Lys Lys Ile Val ThrAla Thr Val Asn 225 230 235 240 Asn Ser Val Leu Gln Lys Gln Gln His AspLeu Met Glu Thr Val Asn 245 250 255 Asn Leu Leu Thr Met Met Ser Thr SerAsn Ser Ala Lys Asp Pro Thr 260 265 270 Val Ala Lys Glu Glu Gln Ile SerPhe Arg Asp Cys Ala Glu Val Phe 275 280 285 Lys Ser Gly His Thr Thr AsnGly Ile Tyr Thr Leu Thr Phe Pro Asn 290 295 300 Ser Thr Glu Glu Ile LysAla Tyr Cys Asp Met Glu Ala Gly Gly Gly 305 310 315 320 Gly Trp Thr IleIle Gln Arg Arg Glu Asp Gly Ser Val Asp Phe Gln 325 330 335 Arg Thr TrpLys Glu Tyr Lys Val Gly Phe Gly Asn Pro Ser Gly Glu 340 345 350 Tyr TrpLeu Gly Asn Glu Phe Val Ser Gln Leu Thr Asn Gln Gln Arg 355 360 365 TyrVal Leu Lys Ile His Leu Lys Asp Trp Glu Gly Asn Glu Ala Tyr 370 375 380Ser Leu Tyr Glu His Phe Tyr Leu Ser Ser Glu Glu Leu Asn Tyr Arg 385 390395 400 Ile His Leu Lys Gly Leu Thr Gly Thr Ala Gly Lys Ile Ser Ser Ile405 410 415 Ser Gln Pro Gly Asn Asp Phe Ser Thr Lys Asp Gly Asp Asn AspLys 420 425 430 Cys Ile Cys Lys Cys Ser Gln Met Leu Thr Gly Gly Trp TrpPhe Asp 435 440 445 Ala Cys Gly Pro Ser Asn Leu Asn Gly Met Tyr Tyr ProGln Arg Gln 450 455 460 Asn Thr Asn Lys Phe Asn Gly Ile Lys Trp Tyr TyrTrp Lys Gly Ser 465 470 475 480 Gly Tyr Ser Leu Lys Ala Thr Thr Met MetIle Arg Pro Ala Asp Phe 485 490 495 7 478 PRT Homo sapiens 7 Asn Gln ArgArg Ser Pro Glu Asn Ser Gly Arg Arg Tyr Asn Arg Ile 1 5 10 15 Gln HisGly Gln Cys Ala Tyr Thr Phe Ile Leu Pro Glu His Asp Gly 20 25 30 Asn CysArg Glu Ser Thr Thr Asp Gln Tyr Asn Thr Asn Ala Leu Gln 35 40 45 Arg AspAla Pro His Val Glu Pro Asp Phe Ser Ser Gln Lys Leu Gln 50 55 60 His LeuGlu His Val Met Glu Asn Tyr Thr Gln Trp Leu Gln Lys Leu 65 70 75 80 GluAsn Tyr Ile Val Glu Asn Met Lys Ser Glu Met Ala Gln Ile Gln 85 90 95 GlnAsn Ala Val Gln Asn His Thr Ala Thr Met Leu Glu Ile Gly Thr 100 105 110Ser Leu Leu Ser Gln Thr Ala Glu Gln Thr Arg Lys Leu Thr Asp Val 115 120125 Glu Thr Gln Val Leu Asn Gln Thr Ser Arg Leu Glu Ile Gln Leu Leu 130135 140 Glu Asn Ser Leu Ser Thr Tyr Lys Leu Glu Lys Gln Leu Leu Gln Gln145 150 155 160 Thr Asn Glu Ile Leu Lys Ile His Glu Lys Asn Ser Leu LeuGlu His 165 170 175 Lys Ile Leu Glu Met Glu Gly Lys His Lys Glu Glu LeuAsp Thr Leu 180 185 190 Lys Glu Glu Lys Glu Asn Leu Gln Gly Leu Val ThrArg Gln Thr Tyr 195 200 205 Ile Ile Gln Glu Leu Glu Lys Gln Leu Asn ArgAla Thr Thr Asn Asn 210 215 220 Ser Val Leu Gln Lys Gln Gln Leu Glu LeuMet Asp Thr Val His Asn 225 230 235 240 Leu Val Asn Leu Cys Thr Lys GluGly Val Leu Leu Lys Gly Gly Lys 245 250 255 Arg Glu Glu Glu Lys Pro PheArg Asp Cys Ala Asp Val Tyr Gln Ala 260 265 270 Gly Phe Asn Lys Ser GlyIle Tyr Thr Ile Tyr Ile Asn Asn Met Pro 275 280 285 Glu Pro Lys Lys ValPhe Cys Asn Met Asp Val Asn Gly Gly Gly Trp 290 295 300 Thr Val Ile GlnHis Arg Glu Asp Gly Ser Leu Asp Phe Gln Arg Gly 305 310 315 320 Trp LysGlu Tyr Lys Met Gly Phe Gly Asn Pro Ser Gly Glu Tyr Trp 325 330 335 LeuGly Asn Glu Phe Ile Phe Ala Ile Thr Ser Gln Arg Gln Tyr Met 340 345 350Leu Arg Ile Glu Leu Met Asp Trp Glu Gly Asn Arg Ala Tyr Ser Gln 355 360365 Tyr Asp Arg Phe His Ile Gly Asn Glu Lys Gln Asn Tyr Arg Leu Tyr 370375 380 Leu Lys Gly His Thr Gly Thr Ala Gly Lys Gln Ser Ser Leu Ile Leu385 390 395 400 His Gly Ala Asp Phe Ser Thr Lys Asp Ala Asp Asn Asp AsnCys Met 405 410 415 Cys Lys Cys Ala Leu Met Leu Thr Gly Gly Trp Trp PheAsp Ala Cys 420 425 430 Gly Pro Ser Asn Leu Asn Gly Met Phe Tyr Thr AlaGly Gln Asn His 435 440 445 Gly Lys Leu Asn Gly Ile Lys Trp His Tyr PheLys Gly Pro Ser Tyr 450 455 460 Ser Leu Arg Ser Thr Thr Met Met Ile ArgPro Leu Asp Phe 465 470 475 8 480 PRT Homo sapiens 8 Ala Ala Tyr Asn AsnPhe Arg Lys Ser Met Asp Ser Ile Gly Lys Lys 1 5 10 15 Gln Tyr Gln ValGln His Gly Ser Cys Ser Tyr Thr Phe Leu Leu Pro 20 25 30 Glu Met Asp AsnCys Arg Ser Ser Ser Ser Pro Tyr Val Ser Asn Ala 35 40 45 Val Gln Arg AspAla Pro Leu Glu Tyr Asp Asp Ser Val Gln Arg Leu 50 55 60 Gln Val Leu GluAsn Ile Met Glu Asn Asn Thr Gln Trp Leu Met Lys 65 70 75 80 Leu Glu AsnTyr Ile Gln Asp Asn Met Lys Lys Glu Met Val Glu Ile 85 90 95 Gln Gln AsnAla Val Gln Asn Gln Thr Ala Val Met Ile Glu Ile Gly 100 105 110 Thr AsnLeu Leu Asn Gln Thr Ala Glu Gln Thr Arg Lys Leu Thr Asp 115 120 125 ValGlu Ala Gln Val Leu Asn Gln Thr Thr Arg Leu Glu Leu Gln Leu 130 135 140Leu Glu His Ser Leu Ser Thr Asn Lys Leu Glu Lys Gln Ile Leu Asp 145 150155 160 Gln Thr Ser Glu Ile Asn Lys Leu Gln Asp Lys Asn Ser Phe Leu Glu165 170 175 Lys Lys Val Leu Ala Met Glu Asp Lys His Ile Ile Gln Leu GlnSer 180 185 190 Ile Lys Glu Glu Lys Asp Gln Leu Gln Val Leu Val Ser LysGln Asn 195 200 205 Ser Ile Ile Glu Glu Leu Glu Lys Lys Ile Val Thr AlaThr Val Asn 210 215 220 Asn Ser Val Leu Gln Lys Gln Gln His Asp Leu MetGlu Thr Val Asn 225 230 235 240 Asn Leu Leu Thr Met Met Ser Thr Ser AsnSer Ala Lys Asp Pro Thr 245 250 255 Val Ala Lys Glu Glu Gln Ile Ser PheArg Asp Cys Ala Glu Val Phe 260 265 270 Lys Ser Gly His Thr Thr Asn GlyIle Tyr Thr Leu Thr Phe Pro Asn 275 280 285 Ser Thr Glu Glu Ile Lys AlaTyr Cys Asp Met Glu Ala Gly Gly Gly 290 295 300 Gly Trp Thr Ile Ile GlnArg Arg Glu Asp Gly Ser Val Asp Phe Gln 305 310 315 320 Arg Thr Trp LysGlu Tyr Lys Val Gly Phe Gly Asn Pro Ser Gly Glu 325 330 335 Tyr Trp LeuGly Asn Glu Phe Val Ser Gln Leu Thr Asn Gln Gln Arg 340 345 350 Tyr ValLeu Lys Ile His Leu Lys Asp Trp Glu Gly Asn Glu Ala Tyr 355 360 365 SerLeu Tyr Glu His Phe Tyr Leu Ser Ser Glu Glu Leu Asn Tyr Arg 370 375 380Ile His Leu Lys Gly Leu Thr Gly Thr Ala Gly Lys Ile Ser Ser Ile 385 390395 400 Ser Gln Pro Gly Asn Asp Phe Ser Thr Lys Asp Gly Asp Asn Asp Lys405 410 415 Cys Ile Cys Lys Cys Ser Gln Met Leu Thr Gly Gly Trp Trp PheAsp 420 425 430 Ala Cys Gly Pro Ser Asn Leu Asn Gly Met Tyr Tyr Pro GlnArg Gln 435 440 445 Asn Thr Asn Lys Phe Asn Gly Ile Lys Trp Tyr Tyr TrpLys Gly Ser 450 455 460 Gly Tyr Ser Leu Lys Ala Thr Thr Met Met Ile ArgPro Ala Asp Phe 465 470 475 480 9 1849 DNA Homo sapiens CDS (47)..(1573)The fibrinogen-like domain starts at position 929. 9 ctgtcctggtacctgacaag accacctcac caccacttgg tctcag atg ctc tgc 55 Met Leu Cys 1 cagcca gct atg cta cta gat ggc ctc ctc ctg ctg gcc acc atg gct 103 Gln ProAla Met Leu Leu Asp Gly Leu Leu Leu Leu Ala Thr Met Ala 5 10 15 gca gcccag cac aga ggg cca gaa gcc ggt ggg cac cgc cag att cac 151 Ala Ala GlnHis Arg Gly Pro Glu Ala Gly Gly His Arg Gln Ile His 20 25 30 35 cag gtccgg cgt ggc cag tgc agc tac acc ttt gtg gtg ccg gag cct 199 Gln Val ArgArg Gly Gln Cys Ser Tyr Thr Phe Val Val Pro Glu Pro 40 45 50 gat atc tgccag ctg gcg ccg aca gcg gcg cct gag gct ttg ggg ggc 247 Asp Ile Cys GlnLeu Ala Pro Thr Ala Ala Pro Glu Ala Leu Gly Gly 55 60 65 tcc aat agc ctccag agg gac ttg cct gcc tcg agg ctg cac cta aca 295 Ser Asn Ser Leu GlnArg Asp Leu Pro Ala Ser Arg Leu His Leu Thr 70 75 80 gac tgg cga gcc cagagg gcc cag cgg gcc cag cgt gtg agc cag ctg 343 Asp Trp Arg Ala Gln ArgAla Gln Arg Ala Gln Arg Val Ser Gln Leu 85 90 95 gag aag ata cta gag aataac act cag tgg ctg ctg aag ctg gag cag 391 Glu Lys Ile Leu Glu Asn AsnThr Gln Trp Leu Leu Lys Leu Glu Gln 100 105 110 115 tcc atc aag gtg aacttg agg tca cac ctg gtg cag gcc cag cag gac 439 Ser Ile Lys Val Asn LeuArg Ser His Leu Val Gln Ala Gln Gln Asp 120 125 130 aca atc cag aac cagaca act acc atg ctg gca ctg ggt gcc aac ctc 487 Thr Ile Gln Asn Gln ThrThr Thr Met Leu Ala Leu Gly Ala Asn Leu 135 140 145 atg aac cag acc aaagct cag acc cac aag ctg act gct gtg gag gca 535 Met Asn Gln Thr Lys AlaGln Thr His Lys Leu Thr Ala Val Glu Ala 150 155 160 cag gtc cta aac cagaca ttg cac atg aag acc caa atg ctg gag aac 583 Gln Val Leu Asn Gln ThrLeu His Met Lys Thr Gln Met Leu Glu Asn 165 170 175 tca ctg tcc acc aacaag ctg gag cgg cag atg ctg atg cag agc cga 631 Ser Leu Ser Thr Asn LysLeu Glu Arg Gln Met Leu Met Gln Ser Arg 180 185 190 195 gag ctg cag cggctg cag ggt cgc aac agg gcc ctg gag acc agg ctg 679 Glu Leu Gln Arg LeuGln Gly Arg Asn Arg Ala Leu Glu Thr Arg Leu 200 205 210 cag gca ctg gaagca caa cat cag gcc cag ctt aac agc ctc caa gag 727 Gln Ala Leu Glu AlaGln His Gln Ala Gln Leu Asn Ser Leu Gln Glu 215 220 225 aag agg gaa caactg cac agt ctc ctg ggc cat cag acc ggg acc ctg 775 Lys Arg Glu Gln LeuHis Ser Leu Leu Gly His Gln Thr Gly Thr Leu 230 235 240 gct aac ctg aagcac aat ctg cac gct ctc agc agc aat tcc agc tcc 823 Ala Asn Leu Lys HisAsn Leu His Ala Leu Ser Ser Asn Ser Ser Ser 245 250 255 ctg cag cag cagcag cag caa ctg acg gag ttt gta cag cgc ctg gta 871 Leu Gln Gln Gln GlnGln Gln Leu Thr Glu Phe Val Gln Arg Leu Val 260 265 270 275 cgg att gtagcc cag gac cag cat ccg gtt tcc tta aag aca cct aag 919 Arg Ile Val AlaGln Asp Gln His Pro Val Ser Leu Lys Thr Pro Lys 280 285 290 cca gtg ttccag gac tgt gca gag atc aag cgc tcc ggg gtt aat acc 967 Pro Val Phe GlnAsp Cys Ala Glu Ile Lys Arg Ser Gly Val Asn Thr 295 300 305 agc ggt gtctat acc atc tat gag acc aac atg aca aag cct ctc aag 1015 Ser Gly Val TyrThr Ile Tyr Glu Thr Asn Met Thr Lys Pro Leu Lys 310 315 320 gtg ttc tgtgac atg gag act gat gga ggt ggc tgg acc ctc atc cag 1063 Val Phe Cys AspMet Glu Thr Asp Gly Gly Gly Trp Thr Leu Ile Gln 325 330 335 cac cgg gaggat gga agc gta aat ttc cag agg acc tgg gaa gaa tac 1111 His Arg Glu AspGly Ser Val Asn Phe Gln Arg Thr Trp Glu Glu Tyr 340 345 350 355 aaa gagggt ttt ggt aat gtg gcc aga gag cac tgg ctg ggc aat gag 1159 Lys Glu GlyPhe Gly Asn Val Ala Arg Glu His Trp Leu Gly Asn Glu 360 365 370 gct gtgcac cgc ctc acc agc aga acg gcc tac ttg cta cgc gtg gaa 1207 Ala Val HisArg Leu Thr Ser Arg Thr Ala Tyr Leu Leu Arg Val Glu 375 380 385 ctg catgac tgg gaa ggc cgc cag acc tcc atc cag tat gag aac ttc 1255 Leu His AspTrp Glu Gly Arg Gln Thr Ser Ile Gln Tyr Glu Asn Phe 390 395 400 cag ctgggc agc gag agg cag cgg tac agc ctc tct gtg aat gac agc 1303 Gln Leu GlySer Glu Arg Gln Arg Tyr Ser Leu Ser Val Asn Asp Ser 405 410 415 agc agttca gca ggg cgc aag aac agc ctg gct cct cag ggc acc aag 1351 Ser Ser SerAla Gly Arg Lys Asn Ser Leu Ala Pro Gln Gly Thr Lys 420 425 430 435 ttcagc acc aaa gac atg gac aat gat aac tgc atg tgt aaa tgt gct 1399 Phe SerThr Lys Asp Met Asp Asn Asp Asn Cys Met Cys Lys Cys Ala 440 445 450 cagatg ctg tct gga ggg tgg tgg ttt gat gcc tgt ggc ctc tcc aac 1447 Gln MetLeu Ser Gly Gly Trp Trp Phe Asp Ala Cys Gly Leu Ser Asn 455 460 465 ctcaat ggc atc tac tat tca gtt cat cag cac ttg cac aag atc aat 1495 Leu AsnGly Ile Tyr Tyr Ser Val His Gln His Leu His Lys Ile Asn 470 475 480 ggcatc cgc tgg cac tac ttc cga ggc ccc agc tac tca ctg cac ggc 1543 Gly IleArg Trp His Tyr Phe Arg Gly Pro Ser Tyr Ser Leu His Gly 485 490 495 acacgc atg atg ctg agg cca atg ggt gcc tgacacacag ccctgcagag 1593 Thr ArgMet Met Leu Arg Pro Met Gly Ala 500 505 actgatgccg taggaggatt ctcaacccaggtgactctgt gcacgctggg ccctgcccag 1653 aaatcagtgc ccagggctca tcttgacattctggaacatc ggaaccagct taccttgccc 1713 ctgaattaca agaattcacc tgcctccctgttgccctcta attgtgaaat tgctgggtgc 1773 ttgaaggcac ctgcctctgt tggaaccatactctttcccc ctcctgctgc atgcccggga 1833 atccctgcca tgaact 1849 10 509 PRTHomo sapiens 10 Met Leu Cys Gln Pro Ala Met Leu Leu Asp Gly Leu Leu LeuLeu Ala 1 5 10 15 Thr Met Ala Ala Ala Gln His Arg Gly Pro Glu Ala GlyGly His Arg 20 25 30 Gln Ile His Gln Val Arg Arg Gly Gln Cys Ser Tyr ThrPhe Val Val 35 40 45 Pro Glu Pro Asp Ile Cys Gln Leu Ala Pro Thr Ala AlaPro Glu Ala 50 55 60 Leu Gly Gly Ser Asn Ser Leu Gln Arg Asp Leu Pro AlaSer Arg Leu 65 70 75 80 His Leu Thr Asp Trp Arg Ala Gln Arg Ala Gln ArgAla Gln Arg Val 85 90 95 Ser Gln Leu Glu Lys Ile Leu Glu Asn Asn Thr GlnTrp Leu Leu Lys 100 105 110 Leu Glu Gln Ser Ile Lys Val Asn Leu Arg SerHis Leu Val Gln Ala 115 120 125 Gln Gln Asp Thr Ile Gln Asn Gln Thr ThrThr Met Leu Ala Leu Gly 130 135 140 Ala Asn Leu Met Asn Gln Thr Lys AlaGln Thr His Lys Leu Thr Ala 145 150 155 160 Val Glu Ala Gln Val Leu AsnGln Thr Leu His Met Lys Thr Gln Met 165 170 175 Leu Glu Asn Ser Leu SerThr Asn Lys Leu Glu Arg Gln Met Leu Met 180 185 190 Gln Ser Arg Glu LeuGln Arg Leu Gln Gly Arg Asn Arg Ala Leu Glu 195 200 205 Thr Arg Leu GlnAla Leu Glu Ala Gln His Gln Ala Gln Leu Asn Ser 210 215 220 Leu Gln GluLys Arg Glu Gln Leu His Ser Leu Leu Gly His Gln Thr 225 230 235 240 GlyThr Leu Ala Asn Leu Lys His Asn Leu His Ala Leu Ser Ser Asn 245 250 255Ser Ser Ser Leu Gln Gln Gln Gln Gln Gln Leu Thr Glu Phe Val Gln 260 265270 Arg Leu Val Arg Ile Val Ala Gln Asp Gln His Pro Val Ser Leu Lys 275280 285 Thr Pro Lys Pro Val Phe Gln Asp Cys Ala Glu Ile Lys Arg Ser Gly290 295 300 Val Asn Thr Ser Gly Val Tyr Thr Ile Tyr Glu Thr Asn Met ThrLys 305 310 315 320 Pro Leu Lys Val Phe Cys Asp Met Glu Thr Asp Gly GlyGly Trp Thr 325 330 335 Leu Ile Gln His Arg Glu Asp Gly Ser Val Asn PheGln Arg Thr Trp 340 345 350 Glu Glu Tyr Lys Glu Gly Phe Gly Asn Val AlaArg Glu His Trp Leu 355 360 365 Gly Asn Glu Ala Val His Arg Leu Thr SerArg Thr Ala Tyr Leu Leu 370 375 380 Arg Val Glu Leu His Asp Trp Glu GlyArg Gln Thr Ser Ile Gln Tyr 385 390 395 400 Glu Asn Phe Gln Leu Gly SerGlu Arg Gln Arg Tyr Ser Leu Ser Val 405 410 415 Asn Asp Ser Ser Ser SerAla Gly Arg Lys Asn Ser Leu Ala Pro Gln 420 425 430 Gly Thr Lys Phe SerThr Lys Asp Met Asp Asn Asp Asn Cys Met Cys 435 440 445 Lys Cys Ala GlnMet Leu Ser Gly Gly Trp Trp Phe Asp Ala Cys Gly 450 455 460 Leu Ser AsnLeu Asn Gly Ile Tyr Tyr Ser Val His Gln His Leu His 465 470 475 480 LysIle Asn Gly Ile Arg Trp His Tyr Phe Arg Gly Pro Ser Tyr Ser 485 490 495Leu His Gly Thr Arg Met Met Leu Arg Pro Met Gly Ala 500 505 11 503 PRTHomo sapiens 11 Met Leu Leu Asp Gly Leu Leu Leu Leu Ala Thr Met Ala AlaAla Gln 1 5 10 15 His Arg Gly Pro Glu Ala Gly Gly His Arg Gln Ile HisGln Val Arg 20 25 30 Arg Gly Gln Cys Ser Tyr Thr Phe Val Val Pro Glu ProAsp Ile Cys 35 40 45 Gln Leu Ala Pro Thr Ala Ala Pro Glu Ala Leu Gly GlySer Asn Ser 50 55 60 Leu Gln Arg Asp Leu Pro Ala Ser Arg Leu His Leu ThrAsp Trp Arg 65 70 75 80 Ala Gln Arg Ala Gln Arg Ala Gln Arg Val Ser GlnLeu Glu Lys Ile 85 90 95 Leu Glu Asn Asn Thr Gln Trp Leu Leu Lys Leu GluGln Ser Ile Lys 100 105 110 Val Asn Leu Arg Ser His Leu Val Gln Ala GlnGln Asp Thr Ile Gln 115 120 125 Asn Gln Thr Thr Thr Met Leu Ala Leu GlyAla Asn Leu Met Asn Gln 130 135 140 Thr Lys Ala Gln Thr His Lys Leu ThrAla Val Glu Ala Gln Val Leu 145 150 155 160 Asn Gln Thr Leu His Met LysThr Gln Met Leu Glu Asn Ser Leu Ser 165 170 175 Thr Asn Lys Leu Glu ArgGln Met Leu Met Gln Ser Arg Glu Leu Gln 180 185 190 Arg Leu Gln Gly ArgAsn Arg Ala Leu Glu Thr Arg Leu Gln Ala Leu 195 200 205 Glu Ala Gln HisGln Ala Gln Leu Asn Ser Leu Gln Glu Lys Arg Glu 210 215 220 Gln Leu HisSer Leu Leu Gly His Gln Thr Gly Thr Leu Ala Asn Leu 225 230 235 240 LysHis Asn Leu His Ala Leu Ser Ser Asn Ser Ser Ser Leu Gln Gln 245 250 255Gln Gln Gln Gln Leu Thr Glu Phe Val Gln Arg Leu Val Arg Ile Val 260 265270 Ala Gln Asp Gln His Pro Val Ser Leu Lys Thr Pro Lys Pro Val Phe 275280 285 Gln Asp Cys Ala Glu Ile Lys Arg Ser Gly Val Asn Thr Ser Gly Val290 295 300 Tyr Thr Ile Tyr Glu Thr Asn Met Thr Lys Pro Leu Lys Val PheCys 305 310 315 320 Asp Met Glu Thr Asp Gly Gly Gly Trp Thr Leu Ile GlnHis Arg Glu 325 330 335 Asp Gly Ser Val Asn Phe Gln Arg Thr Trp Glu GluTyr Lys Glu Gly 340 345 350 Phe Gly Asn Val Ala Arg Glu His Trp Leu GlyAsn Glu Ala Val His 355 360 365 Arg Leu Thr Ser Arg Thr Ala Tyr Leu LeuArg Val Glu Leu His Asp 370 375 380 Trp Glu Gly Arg Gln Thr Ser Ile GlnTyr Glu Asn Phe Gln Leu Gly 385 390 395 400 Ser Glu Arg Gln Arg Tyr SerLeu Ser Val Asn Asp Ser Ser Ser Ser 405 410 415 Ala Gly Arg Lys Asn SerLeu Ala Pro Gln Gly Thr Lys Phe Ser Thr 420 425 430 Lys Asp Met Asp AsnAsp Asn Cys Met Cys Lys Cys Ala Gln Met Leu 435 440 445 Ser Gly Gly TrpTrp Phe Asp Ala Cys Gly Leu Ser Asn Leu Asn Gly 450 455 460 Ile Tyr TyrSer Val His Gln His Leu His Lys Ile Asn Gly Ile Arg 465 470 475 480 TrpHis Tyr Phe Arg Gly Pro Ser Tyr Ser Ile His Gly Thr Arg Met 485 490 495Met Leu Arg Pro Met Gly Ala 500 12 490 PRT Homo sapiens 12 Ala Phe LeuAla Ala Ile Leu Thr His Ile Gly Cys Ser Asn Gln Arg 1 5 10 15 Arg SerPro Glu Asn Ser Gly Arg Arg Tyr Asn Arg Ile Gln His Gly 20 25 30 Gln CysAla Tyr Thr Phe Ile Leu Pro Glu His Asp Gly Asn Cys Arg 35 40 45 Glu SerThr Thr Asp Gln Tyr Asn Thr Asn Ala Leu Gln Arg Asp Ala 50 55 60 Pro HisVal Glu Pro Asp Phe Ser Ser Gln Lys Leu Gln His Leu Glu 65 70 75 80 HisVal Met Glu Asn Tyr Thr Gln Trp Leu Gln Lys Leu Glu Asn Tyr 85 90 95 IleVal Glu Asn Met Lys Ser Glu Met Ala Gln Ile Gln Gln Asn Ala 100 105 110Val Gln Asn His Thr Ala Thr Met Leu Glu Ile Gly Thr Ser Leu Leu 115 120125 Ser Gln Thr Ala Glu Gln Thr Arg Lys Leu Thr Asp Val Glu Thr Gln 130135 140 Val Leu Asn Gln Thr Ser Arg Leu Glu Ile Gln Leu Leu Glu Asn Ser145 150 155 160 Leu Ser Thr Tyr Lys Leu Glu Lys Gln Leu Leu Gln Gln ThrAsn Glu 165 170 175 Ile Leu Lys Ile His Glu Lys Asn Ser Leu Leu Glu HisLys Ile Leu 180 185 190 Glu Met Glu Gly Lys His Lys Glu Glu Leu Asp ThrLeu Lys Glu Glu 195 200 205 Lys Glu Asn Leu Gln Gly Leu Val Thr Arg GlnThr Tyr Ile Ile Gln 210 215 220 Glu Leu Glu Lys Gln Leu Asn Arg Ala ThrThr Asn Asn Ser Val Leu 225 230 235 240 Gln Lys Gln Gln Leu Glu Leu MetAsp Thr Val His Asn Leu Val Asn 245 250 255 Leu Cys Thr Lys Glu Val LeuLeu Lys Gly Gly Lys Arg Glu Glu Glu 260 265 270 Lys Pro Phe Arg Asp CysAla Asp Val Tyr Gln Ala Gly Phe Asn Lys 275 280 285 Ser Gly Ile Tyr ThrIle Tyr Ile Asn Asn Met Pro Glu Pro Lys Lys 290 295 300 Val Phe Cys AsnMet Asp Val Asn Gly Gly Gly Trp Thr Val Ile Gln 305 310 315 320 His ArgGlu Asp Gly Ser Leu Asp Phe Gln Arg Gly Trp Lys Glu Tyr 325 330 335 LysMet Gly Phe Gly Asn Pro Ser Gly Glu Tyr Trp Leu Gly Asn Glu 340 345 350Phe Ile Phe Ala Ile Thr Ser Gln Arg Gln Tyr Met Leu Arg Ile Glu 355 360365 Leu Met Asp Trp Glu Gly Asn Arg Ala Tyr Ser Gln Tyr Asp Arg Phe 370375 380 His Ile Gly Asn Glu Lys Gln Asn Tyr Arg Leu Tyr Leu Lys Gly His385 390 395 400 Thr Gly Thr Ala Gly Lys Gln Ser Ser Leu Ile Leu His GlyAla Asp 405 410 415 Phe Ser Thr Lys Asp Ala Asp Asn Asp Asn Cys Met CysLys Cys Ala 420 425 430 Leu Met Leu Thr Gly Gly Trp Trp Phe Asp Ala CysGly Pro Ser Asn 435 440 445 Leu Asn Gly Met Phe Tyr Thr Ala Gly Gln AsnHis Gly Lys Leu Asn 450 455 460 Gly Ile Lys Trp His Tyr Phe Lys Gly ProSer Tyr Ser Ile Arg Ser 465 470 475 480 Thr Thr Met Met Ile Arg Pro LeuAsp Phe 485 490 13 491 PRT Homo sapiens 13 Ala Phe Leu Ala Ala Ile LeuAla His Ile Gly Cys Thr Thr Gln Arg 1 5 10 15 Arg Ser Pro Glu Asn SerGly Arg Arg Phe Asn Arg Ile Gln His Gly 20 25 30 Gln Cys Thr Tyr Thr PheIle Leu Pro Glu Gln Asp Gly Asn Cys Arg 35 40 45 Glu Ser Thr Thr Asp GlnTyr Asn Thr Asn Ala Leu Gln Arg Asp Ala 50 55 60 Pro His Val Glu Gln AspPhe Ser Phe Gln Lys Leu Gln His Leu Glu 65 70 75 80 His Val Met Glu AsnTyr Thr Gln Trp Leu Gln Lys Leu Glu Ser Tyr 85 90 95 Ile Val Glu Asn MetLys Ser Glu Met Ala Gln Leu Gln Gln Asn Ala 100 105 110 Val Gln Asn HisThr Ala Thr Met Leu Glu Ile Gly Thr Ser Leu Leu 115 120 125 Ser Gln ThrAla Glu Gln Thr Arg Lys Leu Thr Asp Val Glu Thr Gln 130 135 140 Val LeuAsn Gln Thr Ser Arg Leu Glu Ile Gln Leu Leu Glu Asn Ser 145 150 155 160Leu Ser Thr Tyr Lys Leu Glu Lys Gln Leu Leu Gln Gln Thr Asn Glu 165 170175 Ile Leu Lys Ile His Glu Lys Asn Ser Leu Leu Glu His Lys Ile Leu 180185 190 Glu Met Glu Glu Arg His Lys Glu Glu Met Asp Thr Leu Lys Glu Glu195 200 205 Lys Glu Asn Leu Gln Gly Leu Val Thr Arg Gln Ser Tyr Ile IleGln 210 215 220 Glu Leu Glu Lys Gln Leu Asn Lys Ala Thr Thr Asn Asn SerVal Leu 225 230 235 240 Gln Lys Gln Gln Leu Glu Leu Met Asp Thr Val HisThr Leu Ile Thr 245 250 255 Leu Cys Ser Lys Glu Gly Val Leu Leu Lys AsnAla Lys Arg Glu Glu 260 265 270 Glu Lys Pro Phe Arg Asp Cys Ala Asp ValTyr Gln Ala Gly Phe Asn 275 280 285 Lys Ser Gly Ile Tyr Thr Ile Tyr IleAsn Asn Val Ser Asp Pro Lys 290 295 300 Lys Val Phe Cys Asn Met Asp ValAsn Gly Gly Gly Trp Thr Val Ile 305 310 315 320 Gln His Arg Glu Asp GlySer Leu Asp Phe Gln Lys Gly Trp Lys Glu 325 330 335 Tyr Lys Met Gly PheGly Ser Pro Ser Gly Glu Tyr Trp Leu Gly Asn 340 345 350 Glu Phe Ile PheAla Ile Thr Ser Gln Arg Gln Tyr Ser Leu Arg Ile 355 360 365 Glu Leu MetAsp Trp Glu Gly Asn Arg Ala Tyr Ser Gln Tyr Asp Arg 370 375 380 Phe HisIle Gly Asn Glu Lys Gln Asn Tyr Arg Leu Tyr Leu Lys Gly 385 390 395 400His Ser Gly Thr Ala Gly Lys Gln Ser Ser Leu Ile Leu His Gly Ala 405 410415 Glu Phe Ser Thr Lys Asp Ala Asp Asn Asp Asn Cys Met Cys Lys Cys 420425 430 Ala Leu Met Leu Thr Gly Gly Trp Trp Phe Asp Ala Cys Gly Pro Ser435 440 445 Asn Leu Asn Gly Met Phe Tyr Thr Ala Gly Gln Asn His Gly LysLeu 450 455 460 Asn Gly Ile Lys Trp His Tyr Phe Lys Gly Pro Arg Tyr SerIle Arg 465 470 475 480 Ser Thr Thr Met Met Ile Arg Pro Leu Asp Phe 485490 14 497 PRT Mus sp. 14 Met Thr Val Phe Leu Ser Phe Ala Phe Phe AlaAla Ile Leu Thr His 1 5 10 15 Ile Gly Cys Ser Asn Gln Arg Arg Asn ProGlu Asn Ser Gly Arg Arg 20 25 30 Tyr Asn Arg Ile Gln His Gly Gln Cys AlaTyr Thr Phe Ile Leu Pro 35 40 45 Glu His Asp Gly Asn Cys Arg Glu Ser ThrThr Asp Gln Tyr Asn Thr 50 55 60 Asn Ala Leu Gln Arg Asp Ala Pro His ValGlu Pro Asp Phe Ser Ser 65 70 75 80 Gln Lys Leu Gln His Leu Glu His ValMet Glu Asn Tyr Thr Gln Trp 85 90 95 Leu Gln Lys Leu Glu Asn Tyr Ile ValGlu Asn Met Lys Ser Glu Met 100 105 110 Ala Gln Ile Gln Gln Asn Ala ValGln Asn His Thr Ala Thr Met Leu 115 120 125 Glu Ile Gly Thr Ser Leu LeuSer Gln Thr Ala Glu Gln Thr Arg Lys 130 135 140 Leu Thr Asp Val Glu ThrGln Val Leu Asn Gln Thr Ser Arg Leu Glu 145 150 155 160 Ile Gln Leu LeuGlu Asn Ser Leu Ser Thr Tyr Lys Leu Glu Lys Gln 165 170 175 Leu Leu GlnThr Asn Glu Ile Leu Lys Ile His Glu Lys Asn Ser Leu 180 185 190 Leu GluHis Lys Ile Leu Glu Met Glu Gly Lys His Lys Glu Glu Met 195 200 205 AspThr Leu Lys Glu Glu Lys Glu Asn Leu Gln Gly Leu Val Ser Arg 210 215 220Gln Ser Phe Ile Ile Gln Glu Leu Glu Lys Gln Leu Ser Arg Ala Thr 225 230235 240 Asn Asn Asn Ser Ile Leu Gln Lys Gln Gln Leu Glu Leu Met Asp Thr245 250 255 Val His Asn Leu Ile Ser Leu Cys Thr Lys Glu Gly Val Leu LeuLys 260 265 270 Gly Gly Lys Arg Glu Glu Glu Lys Pro Phe Arg Asp Cys AlaAsp Val 275 280 285 Tyr Gln Ala Gly Phe Asn Lys Ser Gly Ile Tyr Thr IleTyr Phe Asn 290 295 300 Asn Val Pro Glu Pro Lys Lys Val Phe Cys Asn MetAsp Val Asn Gly 305 310 315 320 Gly Gly Trp Thr Val Ile Gln His Arg GluAsp Gly Ser Leu Asp Phe 325 330 335 Gln Lys Gly Trp Lys Glu Tyr Lys MetGly Phe Gly Ser Pro Ser Gly 340 345 350 Glu Tyr Trp Leu Gly Asn Glu PheIle Phe Ala Ile Thr Ser Gln Arg 355 360 365 Gln Tyr Met Leu Arg Ile GluLeu Met Asp Trp Glu Gly Asn Arg Ala 370 375 380 Tyr Ser Gln Tyr Asp ArgPhe His Ile Gly Asn Glu Lys Gln Asn Tyr 385 390 395 400 Arg Leu Tyr LeuLys Gly His Thr Gly Thr Ala Gly Lys Gln Ser Ser 405 410 415 Leu Ile LeuHis Gly Ala Asp Phe Ser Thr Lys Asp Ala Asp Asn Asp 420 425 430 Asn CysMet Cys Lys Cys Ala Leu Met Leu Thr Gly Gly Trp Trp Phe 435 440 445 AspAla Cys Gly Pro Ser Asn Leu Asn Gly Met Phe Tyr Thr Ala Gly 450 455 460Gln Asn His Gly Lys Leu Asn Gly Ile Lys Trp His Tyr Phe Lys Gly 465 470475 480 Pro Arg Tyr Ser Ile Arg Ser Thr Thr Met Met Ile Arg Pro Leu Asp485 490 495 Phe 15 496 PRT Mus sp. 15 Met Trp Gln Ile Ile Phe Leu ThrPhe Gly Trp Asp Ala Val Leu Thr 1 5 10 15 Ser Ala Tyr Ser Asn Phe ArgLys Ser Val Asp Ser Thr Gly Arg Arg 20 25 30 Arg Tyr Arg Ile Gln Asn GlyPro Cys Ala Tyr Thr Phe Leu Leu Pro 35 40 45 Glu Thr Asp Ser Gly Arg SerSer Ser Ser Thr Tyr Met Thr Asn Ala 50 55 60 Val Gln Arg Asp Ala Pro ProAsp Tyr Glu Asp Ser Val Gln Ser Leu 65 70 75 80 Gln Leu Leu Glu Asn ValMet Glu Asn Tyr Thr Gln Trp Leu Met Lys 85 90 95 Leu Glu Asn Tyr Ile GlnAsp Asn Met Lys Lys Glu Met Ala Glu Ile 100 105 110 Gln Gln Asn Val ValGln Asn His Thr Ala Val Met Ile Glu Ile Gly 115 120 125 Thr Ser Leu LeuSer Gln Thr Ala Glu Gln Thr Arg Lys Leu Thr Asp 130 135 140 Val Glu ThrGln Val Leu Asn Gln Thr Thr Arg Leu Glu Leu Gln Leu 145 150 155 160 LeuGln His Ser Ile Ser Thr Tyr Lys Leu Glu Lys Gln Ile Leu Asp 165 170 175Gln Thr Ser Glu Ile Asn Lys Ile His Asn Lys Asn Ser Phe Leu Glu 180 185190 Gln Lys Val Leu Asp Met Glu Gly Lys His Ser Glu Glu Met Gln Thr 195200 205 Met Lys Glu Gln Lys Asp Glu Leu Gln Val Leu Val Ser Lys Gln Ser210 215 220 Ser Val Ile Asp Glu Leu Glu Lys Lys Leu Val Thr Ala Thr ValAsn 225 230 235 240 Asn Ser Leu Leu Gln Lys Gln Gln His Asp Leu Met AspThr Val Asn 245 250 255 Ser Leu Leu Thr Met Met Ser Ser Pro Asn Ser LysSer Ser Leu Ala 260 265 270 Ile Arg Arg Glu Glu Gln Thr Thr Phe Arg AspCys Ala Asp Val Phe 275 280 285 Lys Ala Gly Leu Thr Lys Ser Gly Ile TyrThr Leu Thr Phe Pro Asn 290 295 300 Ser Pro Glu Glu Ile Lys Ala Tyr CysAsn Met Asp Val Gly Gly Gly 305 310 315 320 Gly Trp Thr Val Ile Gln HisArg Glu Asp Gly Ser Leu Asp Phe Gln 325 330 335 Lys Gly Trp Lys Glu TyrLys Met Gly Phe Gly Asn Pro Leu Gly Glu 340 345 350 Tyr Trp Leu Gly AsnGlu Phe Ile Ser Gln Ile Thr Gly Gln His Arg 355 360 365 Tyr Val Leu LysIle Gln Leu Lys Asp Trp Glu Gly Asn Glu Ala His 370 375 380 Ser Leu TyrAsp His Phe Tyr Ile Ala Gly Glu Glu Ser Asn Tyr Arg 385 390 395 400 IleHis Leu Thr Gly Leu Thr Gly Thr Ala Ala Lys Ile Ser Ser Ile 405 410 415Ser Gln Pro Gly Ser Asp Phe Ser Thr Lys Asp Ser Asp Asn Asp Lys 420 425430 Cys Ile Cys Lys Cys Ser Leu Met Leu Thr Gly Gly Trp Trp Phe Asp 435440 445 Ala Cys Gly Pro Ser Asn Leu Asn Gly Gln Phe Tyr Pro Gln Lys Gln450 455 460 Asn Thr Asn Lys Phe Asn Gly Ile Lys Trp Tyr Tyr Trp Lys GlySer 465 470 475 480 Gly Tyr Ser Ile Lys Ala Thr Thr Met Met Ile Arg ProAla Asp Phe 485 490 495 16 496 PRT Homo sapiens 16 Met Trp Gln Ile ValPhe Phe Thr Leu Ser Cys Asp Ala Val Leu Thr 1 5 10 15 Ala Ala Tyr AsnAsn Phe Arg Lys Ser Met Asp Ser Ile Gly Lys Lys 20 25 30 Arg Tyr Arg IleGln His Gly Ser Cys Ala Tyr Thr Phe Leu Leu Pro 35 40 45 Glu Met Asp AsnGly Arg Ser Ser Ser Ser Thr Tyr Val Thr Asn Ala 50 55 60 Val Gln Arg AspAla Pro Pro Glu Tyr Glu Asp Ser Val Gln Ser Leu 65 70 75 80 Gln Leu LeuGlu Asn Val Met Glu Asn Tyr Thr Gln Trp Leu Met Lys 85 90 95 Leu Glu AsnTyr Ile Gln Asp Asn Met Lys Lys Glu Met Ala Glu Ile 100 105 110 Gln GlnAsn Ala Val Gln Asn His Thr Ala Val Met Ile Glu Ile Gly 115 120 125 ThrSer Leu Leu Ser Gln Thr Ala Glu Gln Thr Arg Lys Leu Thr Asp 130 135 140Val Glu Thr Gln Val Leu Asn Gln Thr Thr Arg Leu Glu Leu Gln Leu 145 150155 160 Leu Gln His Ser Ile Ser Thr Tyr Lys Leu Glu Lys Gln Ile Leu Asp165 170 175 Gln Thr Ser Glu Ile Asn Lys Ile His Asp Lys Asn Ser Phe LeuGlu 180 185 190 Lys Lys Val Leu Asp Met Glu Asp Lys His Ile Ile Glu MetGln Thr 195 200 205 Ile Lys Glu Glu Lys Asp Glu Leu Gln Val Leu Val SerLys Gln Asn 210 215 220 Ser Ile Ile Glu Glu Leu Glu Lys Lys Ile Val ThrAla Thr Val Asn 225 230 235 240 Asn Ser Val Leu Gln Lys Gln Gln His AspLeu Met Asp Thr Val Asn 245 250 255 Asn Leu Leu Thr Met Met Ser Thr SerAsn Ser Ala Lys Asp Ser Thr 260 265 270 Val Ala Arg Glu Glu Gln Ile SerPhe Arg Asp Cys Ala Asp Val Phe 275 280 285 Lys Ala Gly His Thr Lys AsnGly Ile Tyr Thr Leu Thr Phe Pro Asn 290 295 300 Ser Pro Glu Glu Ile LysAla Tyr Cys Asn Met Asp Ala Gly Gly Gly 305 310 315 320 Gly Trp Thr IleIle Gln Arg Arg Glu Asp Gly Ser Leu Asp Phe Gln 325 330 335 Lys Gly TrpLys Glu Tyr Lys Val Gly Phe Gly Ser Pro Ser Gly Glu 340 345 350 Tyr TrpLeu Gly Asn Glu Phe Ile Ser Gln Ile Thr Asn Gln Gln Arg 355 360 365 TyrVal Leu Lys Ile His Leu Lys Asp Trp Glu Gly Asn Glu Ala Tyr 370 375 380Ser Leu Tyr Asp His Phe Tyr Ile Ser Gly Glu Glu Leu Asn Tyr Arg 385 390395 400 Ile His Leu Lys Gly Leu Thr Gly Thr Ala Ala Lys Ile Ser Ser Ile405 410 415 Ser Gln Pro Gly Asn Asp Phe Ser Thr Lys Asp Gly Asp Asn AspLys 420 425 430 Cys Ile Cys Lys Cys Ser Leu Met Leu Thr Gly Gly Trp TrpPhe Asp 435 440 445 Ala Cys Gly Pro Ser Asn Leu Asn Gly Met Phe Tyr ProGln Arg Gln 450 455 460 Asn Thr Asn Lys Phe Asn Gly Ile Lys Trp Tyr TyrTrp Lys Gly Ser 465 470 475 480 Gly Tyr Ser Ile Lys Ala Thr Thr Met MetIle Arg Pro Ala Asp Phe 485 490 495 17 1512 DNA Homo sapiens CDS(1)..(1512) 17 atg ctc tcc cag cta gcc atg ctg cag ggc agc ctc ctc cttgtg gtt 48 Met Leu Ser Gln Leu Ala Met Leu Gln Gly Ser Leu Leu Leu ValVal 1 5 10 15 gcc acc atg tct gtg gct caa cag aca agg cag gag gcg gatagg ggc 96 Ala Thr Met Ser Val Ala Gln Gln Thr Arg Gln Glu Ala Asp ArgGly 20 25 30 tgc gag aca ctt gta gtc cag cac ggc cac tgt agc tac acc ttcttg 144 Cys Glu Thr Leu Val Val Gln His Gly His Cys Ser Tyr Thr Phe Leu35 40 45 ctg ccc aag tct gag ccc tgc cct ccg ggg cct gag gtc tcc agg gac192 Leu Pro Lys Ser Glu Pro Cys Pro Pro Gly Pro Glu Val Ser Arg Asp 5055 60 tcc aac acc ctc cag aga gaa tca ctg gcc aac cca ctg cac ctg ggg240 Ser Asn Thr Leu Gln Arg Glu Ser Leu Ala Asn Pro Leu His Leu Gly 6570 75 80 aag ttg ccc acc cag cag gtg aaa cag ctg gag cag gca ctg cag aac288 Lys Leu Pro Thr Gln Gln Val Lys Gln Leu Glu Gln Ala Leu Gln Asn 8590 95 aac acg cag tgg ctg aag aag cta gag agg gcc atc aag acg atc ttg336 Asn Thr Gln Trp Leu Lys Lys Leu Glu Arg Ala Ile Lys Thr Ile Leu 100105 110 agg tcg aag ctg gag cag gtc cag cag caa atg gcc cag aat cag acg384 Arg Ser Lys Leu Glu Gln Val Gln Gln Gln Met Ala Gln Asn Gln Thr 115120 125 gcc ccc atg cta gag ctg ggc acc agc ctc ctg aac cag acc act gcc432 Ala Pro Met Leu Glu Leu Gly Thr Ser Leu Leu Asn Gln Thr Thr Ala 130135 140 cag atc cgc aag ctg acc gac atg gag gct cag ctc ctg aac cag aca480 Gln Ile Arg Lys Leu Thr Asp Met Glu Ala Gln Leu Leu Asn Gln Thr 145150 155 160 tca aga atg gat gcc cag atg cca gag acc ttt ctg tcc acc aacaag 528 Ser Arg Met Asp Ala Gln Met Pro Glu Thr Phe Leu Ser Thr Asn Lys165 170 175 ctg gag aac cag ctg ctg cta cag agg cag aag ctc cag cag cttcag 576 Leu Glu Asn Gln Leu Leu Leu Gln Arg Gln Lys Leu Gln Gln Leu Gln180 185 190 ggc caa aac agc gcg ctc gag aag cgg ttg cag gcc ctg gag accaag 624 Gly Gln Asn Ser Ala Leu Glu Lys Arg Leu Gln Ala Leu Glu Thr Lys195 200 205 cag cag gag gag ctg gcc agc atc ctc agc aag aag gcg aag ctgctg 672 Gln Gln Glu Glu Leu Ala Ser Ile Leu Ser Lys Lys Ala Lys Leu Leu210 215 220 aac acg ctg agc cgc cag agc gcc gcc ctc acc aac atc gag cgcggc 720 Asn Thr Leu Ser Arg Gln Ser Ala Ala Leu Thr Asn Ile Glu Arg Gly225 230 235 240 ctg cgc ggt gtc agg cac aac tcc agc ctc ctg cag gac cagcag cac 768 Leu Arg Gly Val Arg His Asn Ser Ser Leu Leu Gln Asp Gln GlnHis 245 250 255 agc ctg cgc cag ctg ctg gtg ttg ttg cgg cac ctg gtg caagaa agg 816 Ser Leu Arg Gln Leu Leu Val Leu Leu Arg His Leu Val Gln GluArg 260 265 270 gct aac gcc tcg gcc ccg gcc ttc ata atg gca ggt gag caggtg ttc 864 Ala Asn Ala Ser Ala Pro Ala Phe Ile Met Ala Gly Glu Gln ValPhe 275 280 285 cag gac tgt gca gag atc cag cgc tct ggg gcc agt gcc agtggt gtc 912 Gln Asp Cys Ala Glu Ile Gln Arg Ser Gly Ala Ser Ala Ser GlyVal 290 295 300 tac acc atc cag gtg tcc aat gca acg aag ccc agg aag gtgttc tgt 960 Tyr Thr Ile Gln Val Ser Asn Ala Thr Lys Pro Arg Lys Val PheCys 305 310 315 320 gac ctg cag agc agt gga ggc agg tgg acc ctc atc cagcgc cgt gag 1008 Asp Leu Gln Ser Ser Gly Gly Arg Trp Thr Leu Ile Gln ArgArg Glu 325 330 335 aat ggc acc gtg aat ttt cag cgg aac tgg aag gat tacaaa cag ggc 1056 Asn Gly Thr Val Asn Phe Gln Arg Asn Trp Lys Asp Tyr LysGln Gly 340 345 350 ttc gga gac cca gct ggg gag cac tgg ctg ggc aat gaagtg gtg cac 1104 Phe Gly Asp Pro Ala Gly Glu His Trp Leu Gly Asn Glu ValVal His 355 360 365 cag ctc acc aga agg gca gcc tac tct ctg cgt gtg gagctg caa gac 1152 Gln Leu Thr Arg Arg Ala Ala Tyr Ser Leu Arg Val Glu LeuGln Asp 370 375 380 tgg gaa ggc cac gag gcc tat gcc cag tac gaa cat ttccac ctg ggc 1200 Trp Glu Gly His Glu Ala Tyr Ala Gln Tyr Glu His Phe HisLeu Gly 385 390 395 400 agt gag aac cag cta tac agg ctt tct gtg gtc gggtac agc ggc tca 1248 Ser Glu Asn Gln Leu Tyr Arg Leu Ser Val Val Gly TyrSer Gly Ser 405 410 415 gca ggg cgc cag agc agc ctg gtc ctg cag aac accagc ttt agc acc 1296 Ala Gly Arg Gln Ser Ser Leu Val Leu Gln Asn Thr SerPhe Ser Thr 420 425 430 ctt gac tca gac aac gac cac tgt ctc tgc aag tgtgcc cag gtg atg 1344 Leu Asp Ser Asp Asn Asp His Cys Leu Cys Lys Cys AlaGln Val Met 435 440 445 tct gga ggg tgg tgg ttt gac gcc tgt ggc ctg tcaaac ctc aac ggc 1392 Ser Gly Gly Trp Trp Phe Asp Ala Cys Gly Leu Ser AsnLeu Asn Gly 450 455 460 gtc tac tac cac gct ccc gac aac aag tac aag atggac ggc atc cgc 1440 Val Tyr Tyr His Ala Pro Asp Asn Lys Tyr Lys Met AspGly Ile Arg 465 470 475 480 tgg cac tac ttc aag ggc ccc agc tac tca ctgcgt gcc tct cgc atg 1488 Trp His Tyr Phe Lys Gly Pro Ser Tyr Ser Leu ArgAla Ser Arg Met 485 490 495 atg ata cgg cct ttg gac atc taa 1512 Met IleArg Pro Leu Asp Ile 500 18 503 PRT Homo sapiens 18 Met Leu Ser Gln LeuAla Met Leu Gln Gly Ser Leu Leu Leu Val Val 1 5 10 15 Ala Thr Met SerVal Ala Gln Gln Thr Arg Gln Glu Ala Asp Arg Gly 20 25 30 Cys Glu Thr LeuVal Val Gln His Gly His Cys Ser Tyr Thr Phe Leu 35 40 45 Leu Pro Lys SerGlu Pro Cys Pro Pro Gly Pro Glu Val Ser Arg Asp 50 55 60 Ser Asn Thr LeuGln Arg Glu Ser Leu Ala Asn Pro Leu His Leu Gly 65 70 75 80 Lys Leu ProThr Gln Gln Val Lys Gln Leu Glu Gln Ala Leu Gln Asn 85 90 95 Asn Thr GlnTrp Leu Lys Lys Leu Glu Arg Ala Ile Lys Thr Ile Leu 100 105 110 Arg SerLys Leu Glu Gln Val Gln Gln Gln Met Ala Gln Asn Gln Thr 115 120 125 AlaPro Met Leu Glu Leu Gly Thr Ser Leu Leu Asn Gln Thr Thr Ala 130 135 140Gln Ile Arg Lys Leu Thr Asp Met Glu Ala Gln Leu Leu Asn Gln Thr 145 150155 160 Ser Arg Met Asp Ala Gln Met Pro Glu Thr Phe Leu Ser Thr Asn Lys165 170 175 Leu Glu Asn Gln Leu Leu Leu Gln Arg Gln Lys Leu Gln Gln LeuGln 180 185 190 Gly Gln Asn Ser Ala Leu Glu Lys Arg Leu Gln Ala Leu GluThr Lys 195 200 205 Gln Gln Glu Glu Leu Ala Ser Ile Leu Ser Lys Lys AlaLys Leu Leu 210 215 220 Asn Thr Leu Ser Arg Gln Ser Ala Ala Leu Thr AsnIle Glu Arg Gly 225 230 235 240 Leu Arg Gly Val Arg His Asn Ser Ser LeuLeu Gln Asp Gln Gln His 245 250 255 Ser Leu Arg Gln Leu Leu Val Leu LeuArg His Leu Val Gln Glu Arg 260 265 270 Ala Asn Ala Ser Ala Pro Ala PheIle Met Ala Gly Glu Gln Val Phe 275 280 285 Gln Asp Cys Ala Glu Ile GlnArg Ser Gly Ala Ser Ala Ser Gly Val 290 295 300 Tyr Thr Ile Gln Val SerAsn Ala Thr Lys Pro Arg Lys Val Phe Cys 305 310 315 320 Asp Leu Gln SerSer Gly Gly Arg Trp Thr Leu Ile Gln Arg Arg Glu 325 330 335 Asn Gly ThrVal Asn Phe Gln Arg Asn Trp Lys Asp Tyr Lys Gln Gly 340 345 350 Phe GlyAsp Pro Ala Gly Glu His Trp Leu Gly Asn Glu Val Val His 355 360 365 GlnLeu Thr Arg Arg Ala Ala Tyr Ser Leu Arg Val Glu Leu Gln Asp 370 375 380Trp Glu Gly His Glu Ala Tyr Ala Gln Tyr Glu His Phe His Leu Gly 385 390395 400 Ser Glu Asn Gln Leu Tyr Arg Leu Ser Val Val Gly Tyr Ser Gly Ser405 410 415 Ala Gly Arg Gln Ser Ser Leu Val Leu Gln Asn Thr Ser Phe SerThr 420 425 430 Leu Asp Ser Asp Asn Asp His Cys Leu Cys Lys Cys Ala GlnVal Met 435 440 445 Ser Gly Gly Trp Trp Phe Asp Ala Cys Gly Leu Ser AsnLeu Asn Gly 450 455 460 Val Tyr Tyr His Ala Pro Asp Asn Lys Tyr Lys MetAsp Gly Ile Arg 465 470 475 480 Trp His Tyr Phe Lys Gly Pro Ser Tyr SerLeu Arg Ala Ser Arg Met 485 490 495 Met Ile Arg Pro Leu Asp Ile 500 191497 DNA Artificial Sequence Description of Artificial Sequence Chimeric19 atg aca gtt ttc ctt tcc ttt gct ttc ctc gct gcc att ctg act cac 48Met Thr Val Phe Leu Ser Phe Ala Phe Leu Ala Ala Ile Leu Thr His 1 5 1015 ata ggg tgc agc aat cag cgc cga agt cca gaa aac agt ggg aga aga 96Ile Gly Cys Ser Asn Gln Arg Arg Ser Pro Glu Asn Ser Gly Arg Arg 20 25 30tat aac cgg att caa cat ggg caa tgt gcc tac act ttc att ctt cca 144 TyrAsn Arg Ile Gln His Gly Gln Cys Ala Tyr Thr Phe Ile Leu Pro 35 40 45 gaacac gat ggc aac tgt cgt gag agt acg aca gac cag tac aac aca 192 Glu HisAsp Gly Asn Cys Arg Glu Ser Thr Thr Asp Gln Tyr Asn Thr 50 55 60 aac gctctg cag aga gat gct cca cac gtg gaa ccg gat ttc tct tcc 240 Asn Ala LeuGln Arg Asp Ala Pro His Val Glu Pro Asp Phe Ser Ser 65 70 75 80 cag aaactt caa cat ctg gaa cat gtg atg gaa aat tat act cag tgg 288 Gln Lys LeuGln His Leu Glu His Val Met Glu Asn Tyr Thr Gln Trp 85 90 95 ctg caa aaactt gag aat tac att gtg gaa aac atg aag tcg gag atg 336 Leu Gln Lys LeuGlu Asn Tyr Ile Val Glu Asn Met Lys Ser Glu Met 100 105 110 gcc cag atacag cag aat gca gtt cag aac cac acg gct acc atg ctg 384 Ala Gln Ile GlnGln Asn Ala Val Gln Asn His Thr Ala Thr Met Leu 115 120 125 gag ata ggaacc agc ctc ctc tct cag act gca gag cag acc aga aag 432 Glu Ile Gly ThrSer Leu Leu Ser Gln Thr Ala Glu Gln Thr Arg Lys 130 135 140 ctg aca gatgtt gag acc cag gta cta aat caa act tct cga ctt gag 480 Leu Thr Asp ValGlu Thr Gln Val Leu Asn Gln Thr Ser Arg Leu Glu 145 150 155 160 ata cagctg ctg gag aat tca tta tcc acc tac aag cta gag aag caa 528 Ile Gln LeuLeu Glu Asn Ser Leu Ser Thr Tyr Lys Leu Glu Lys Gln 165 170 175 ctt cttcaa cag aca aat gaa atc ttg aag atc cat gaa aaa aac agt 576 Leu Leu GlnGln Thr Asn Glu Ile Leu Lys Ile His Glu Lys Asn Ser 180 185 190 tta ttagaa cat aaa atc tta gaa atg gaa gga aaa cac aag gaa gag 624 Leu Leu GluHis Lys Ile Leu Glu Met Glu Gly Lys His Lys Glu Glu 195 200 205 ttg gacacc tta aag gaa gag aaa gag aac ctt caa ggc ttg gtt act 672 Leu Asp ThrLeu Lys Glu Glu Lys Glu Asn Leu Gln Gly Leu Val Thr 210 215 220 cgt caaaca tat ata atc cag gag ctg gaa aag caa tta aac aga gct 720 Arg Gln ThrTyr Ile Ile Gln Glu Leu Glu Lys Gln Leu Asn Arg Ala 225 230 235 240 accacc aac aac agt gtc ctt cag aag cag caa ctg gag ctg atg gac 768 Thr ThrAsn Asn Ser Val Leu Gln Lys Gln Gln Leu Glu Leu Met Asp 245 250 255 acagtc cac aac ctt gtc aat ctt tgc act aaa gaa ggt gtt tta cta 816 Thr ValHis Asn Leu Val Asn Leu Cys Thr Lys Glu Gly Val Leu Leu 260 265 270 aaggga gga aaa aga gag gaa gag aaa cca ttt aga gac tgt gct gaa 864 Lys GlyGly Lys Arg Glu Glu Glu Lys Pro Phe Arg Asp Cys Ala Glu 275 280 285 gtattc aaa tca gga cac acc aca aat ggc atc tac acg tta aca ttc 912 Val PheLys Ser Gly His Thr Thr Asn Gly Ile Tyr Thr Leu Thr Phe 290 295 300 cctaat tct aca gaa gag atc aag gcc tac tgt gac atg gaa gct gga 960 Pro AsnSer Thr Glu Glu Ile Lys Ala Tyr Cys Asp Met Glu Ala Gly 305 310 315 320gga ggc ggg tgg aca att att cag cga cgt gag gat ggc agc gtt gat 1008 GlyGly Gly Trp Thr Ile Ile Gln Arg Arg Glu Asp Gly Ser Val Asp 325 330 335ttt cag agg act tgg aaa gaa tat aaa gtg gga ttt ggt aac cct tca 1056 PheGln Arg Thr Trp Lys Glu Tyr Lys Val Gly Phe Gly Asn Pro Ser 340 345 350gga gaa tat tgg ctg gga aat gag ttt gtt tcg caa ctg act aat cag 1104 GlyGlu Tyr Trp Leu Gly Asn Glu Phe Val Ser Gln Leu Thr Asn Gln 355 360 365caa cgc tat gtg ctt aaa ata cac ctt aaa gac tgg gaa ggg aat gag 1152 GlnArg Tyr Val Leu Lys Ile His Leu Lys Asp Trp Glu Gly Asn Glu 370 375 380gct tac tca ttg tat gaa cat ttc tat ctc tca agt gaa gaa ctc aat 1200 AlaTyr Ser Leu Tyr Glu His Phe Tyr Leu Ser Ser Glu Glu Leu Asn 385 390 395400 tat agg att cac ctt aaa gga ctt aca ggg aca gcc ggc aaa ata agc 1248Tyr Arg Ile His Leu Lys Gly Leu Thr Gly Thr Ala Gly Lys Ile Ser 405 410415 agc atc agc caa cca gga aat gat ttt agc aca aag gat gga gac aac 1296Ser Ile Ser Gln Pro Gly Asn Asp Phe Ser Thr Lys Asp Gly Asp Asn 420 425430 gac aaa tgt att tgc aaa tgt tca caa atg cta aca gga ggc tgg tgg 1344Asp Lys Cys Ile Cys Lys Cys Ser Gln Met Leu Thr Gly Gly Trp Trp 435 440445 ttt gat gca tgt ggt cct tcc aac ttg aac gga atg tac tat cca cag 1392Phe Asp Ala Cys Gly Pro Ser Asn Leu Asn Gly Met Tyr Tyr Pro Gln 450 455460 agg cag aac aca aat aag ttc aac ggc att aaa tgg tac tac tgg aaa 1440Arg Gln Asn Thr Asn Lys Phe Asn Gly Ile Lys Trp Tyr Tyr Trp Lys 465 470475 480 ggc tca ggc tat tcg ctc aag gcc aca acc atg atg atc cga cca gca1488 Gly Ser Gly Tyr Ser Leu Lys Ala Thr Thr Met Met Ile Arg Pro Ala 485490 495 gat ttc taa 1497 Asp Phe 20 498 PRT Artificial SequenceDescription of Artificial Sequence Chimeric 20 Met Thr Val Phe Leu SerPhe Ala Phe Leu Ala Ala Ile Leu Thr His 1 5 10 15 Ile Gly Cys Ser AsnGln Arg Arg Ser Pro Glu Asn Ser Gly Arg Arg 20 25 30 Tyr Asn Arg Ile GlnHis Gly Gln Cys Ala Tyr Thr Phe Ile Leu Pro 35 40 45 Glu His Asp Gly AsnCys Arg Glu Ser Thr Thr Asp Gln Tyr Asn Thr 50 55 60 Asn Ala Leu Gln ArgAsp Ala Pro His Val Glu Pro Asp Phe Ser Ser 65 70 75 80 Gln Lys Leu GlnHis Leu Glu His Val Met Glu Asn Tyr Thr Gln Trp 85 90 95 Leu Gln Lys LeuGlu Asn Tyr Ile Val Glu Asn Met Lys Ser Glu Met 100 105 110 Ala Gln IleGln Gln Asn Ala Val Gln Asn His Thr Ala Thr Met Leu 115 120 125 Glu IleGly Thr Ser Leu Leu Ser Gln Thr Ala Glu Gln Thr Arg Lys 130 135 140 LeuThr Asp Val Glu Thr Gln Val Leu Asn Gln Thr Ser Arg Leu Glu 145 150 155160 Ile Gln Leu Leu Glu Asn Ser Leu Ser Thr Tyr Lys Leu Glu Lys Gln 165170 175 Leu Leu Gln Gln Thr Asn Glu Ile Leu Lys Ile His Glu Lys Asn Ser180 185 190 Leu Leu Glu His Lys Ile Leu Glu Met Glu Gly Lys His Lys GluGlu 195 200 205 Leu Asp Thr Leu Lys Glu Glu Lys Glu Asn Leu Gln Gly LeuVal Thr 210 215 220 Arg Gln Thr Tyr Ile Ile Gln Glu Leu Glu Lys Gln LeuAsn Arg Ala 225 230 235 240 Thr Thr Asn Asn Ser Val Leu Gln Lys Gln GlnLeu Glu Leu Met Asp 245 250 255 Thr Val His Asn Leu Val Asn Leu Cys ThrLys Glu Gly Val Leu Leu 260 265 270 Lys Gly Gly Lys Arg Glu Glu Glu LysPro Phe Arg Asp Cys Ala Glu 275 280 285 Val Phe Lys Ser Gly His Thr ThrAsn Gly Ile Tyr Thr Leu Thr Phe 290 295 300 Pro Asn Ser Thr Glu Glu IleLys Ala Tyr Cys Asp Met Glu Ala Gly 305 310 315 320 Gly Gly Gly Trp ThrIle Ile Gln Arg Arg Glu Asp Gly Ser Val Asp 325 330 335 Phe Gln Arg ThrTrp Lys Glu Tyr Lys Val Gly Phe Gly Asn Pro Ser 340 345 350 Gly Glu TyrTrp Leu Gly Asn Glu Phe Val Ser Gln Leu Thr Asn Gln 355 360 365 Gln ArgTyr Val Leu Lys Ile His Leu Lys Asp Trp Glu Gly Asn Glu 370 375 380 AlaTyr Ser Leu Tyr Glu His Phe Tyr Leu Ser Ser Glu Glu Leu Asn 385 390 395400 Tyr Arg Ile His Leu Lys Gly Leu Thr Gly Thr Ala Gly Lys Ile Ser 405410 415 Ser Ile Ser Gln Pro Gly Asn Asp Phe Ser Thr Lys Asp Gly Asp Asn420 425 430 Asp Lys Cys Ile Cys Lys Cys Ser Gln Met Leu Thr Gly Gly TrpTrp 435 440 445 Phe Asp Ala Cys Gly Pro Ser Asn Leu Asn Gly Met Tyr TyrPro Gln 450 455 460 Arg Gln Asn Thr Asn Lys Phe Asn Gly Ile Lys Trp TyrTyr Trp Lys 465 470 475 480 Gly Ser Gly Tyr Ser Leu Lys Ala Thr Thr MetMet Ile Arg Pro Ala 485 490 495 Asp Phe 21 1491 DNA Artificial SequenceDescription of Artificial Sequence Chimeric 21 atg tgg cag att gtt ttcttt act ctg agc tgt gat ctt gtc ttg gcc 48 Met Trp Gln Ile Val Phe PheThr Leu Ser Cys Asp Leu Val Leu Ala 1 5 10 15 gca gcc tat aac aac tttcgg aag agc atg gac agc ata gga aag aag 96 Ala Ala Tyr Asn Asn Phe ArgLys Ser Met Asp Ser Ile Gly Lys Lys 20 25 30 caa tat cag gtc cag cat gggtcc tgc agc tac act ttc ctc ctg cca 144 Gln Tyr Gln Val Gln His Gly SerCys Ser Tyr Thr Phe Leu Leu Pro 35 40 45 gag atg gac aac tgc cgc tct tcctcc agc ccc tac gtg tcc aat gct 192 Glu Met Asp Asn Cys Arg Ser Ser SerSer Pro Tyr Val Ser Asn Ala 50 55 60 gtg cag agg gac gcg ccg ctc gaa tacgat gac tcg gtg cag agg ctg 240 Val Gln Arg Asp Ala Pro Leu Glu Tyr AspAsp Ser Val Gln Arg Leu 65 70 75 80 caa gtg ctg gag aac atc atg gaa aacaac act cag tgg cta atg aag 288 Gln Val Leu Glu Asn Ile Met Glu Asn AsnThr Gln Trp Leu Met Lys 85 90 95 ctt gag aat tat atc cag gac aac atg aagaaa gaa atg gta gag ata 336 Leu Glu Asn Tyr Ile Gln Asp Asn Met Lys LysGlu Met Val Glu Ile 100 105 110 cag cag aat gca gta cag aac cag acg gctgtg atg ata gaa ata ggg 384 Gln Gln Asn Ala Val Gln Asn Gln Thr Ala ValMet Ile Glu Ile Gly 115 120 125 aca aac ctg ttg aac caa aca gct gag caaacg cgg aag tta act gat 432 Thr Asn Leu Leu Asn Gln Thr Ala Glu Gln ThrArg Lys Leu Thr Asp 130 135 140 gtg gaa gcc caa gta tta aat cag acc acgaga ctt gaa ctt cag ctc 480 Val Glu Ala Gln Val Leu Asn Gln Thr Thr ArgLeu Glu Leu Gln Leu 145 150 155 160 ttg gaa cac tcc ctc tcg aca aac aaattg gaa aaa cag att ttg gac 528 Leu Glu His Ser Leu Ser Thr Asn Lys LeuGlu Lys Gln Ile Leu Asp 165 170 175 cag acc agt gaa ata aac aaa ttg caagat aag aac agt ttc cta gaa 576 Gln Thr Ser Glu Ile Asn Lys Leu Gln AspLys Asn Ser Phe Leu Glu 180 185 190 aag aag gtg cta gct atg gaa gac aagcac atc atc caa cta cag tca 624 Lys Lys Val Leu Ala Met Glu Asp Lys HisIle Ile Gln Leu Gln Ser 195 200 205 ata aaa gaa gag aaa gat cag cta caggtg tta gta tcc aag caa aat 672 Ile Lys Glu Glu Lys Asp Gln Leu Gln ValLeu Val Ser Lys Gln Asn 210 215 220 tcc atc att gaa gaa cta gaa aaa aaaata gtg act gcc acg gtg aat 720 Ser Ile Ile Glu Glu Leu Glu Lys Lys IleVal Thr Ala Thr Val Asn 225 230 235 240 aat tca gtt ctt caa aag cag caacat gat ctc atg gag aca gtt aat 768 Asn Ser Val Leu Gln Lys Gln Gln HisAsp Leu Met Glu Thr Val Asn 245 250 255 aac tta ctg act atg atg tcc acatca aac tca gct aag gac ccc act 816 Asn Leu Leu Thr Met Met Ser Thr SerAsn Ser Ala Lys Asp Pro Thr 260 265 270 gtt gct aaa gaa gaa caa atc agcttc aga gac tgt gca gat gta tat 864 Val Ala Lys Glu Glu Gln Ile Ser PheArg Asp Cys Ala Asp Val Tyr 275 280 285 caa gct ggt ttt aat aaa agt ggaatc tac act att tat att aat aat 912 Gln Ala Gly Phe Asn Lys Ser Gly IleTyr Thr Ile Tyr Ile Asn Asn 290 295 300 atg cca gaa ccc aaa aag gtg ttttgc aat atg gat gtc aat ggg gga 960 Met Pro Glu Pro Lys Lys Val Phe CysAsn Met Asp Val Asn Gly Gly 305 310 315 320 ggt tgg act gta ata caa catcgt gaa gat gga agt cta gat ttc caa 1008 Gly Trp Thr Val Ile Gln His ArgGlu Asp Gly Ser Leu Asp Phe Gln 325 330 335 aga ggc tgg aag gaa tat aaaatg ggt ttt gga aat ccc tcc ggt gaa 1056 Arg Gly Trp Lys Glu Tyr Lys MetGly Phe Gly Asn Pro Ser Gly Glu 340 345 350 tat tgg ctg ggg aat gag tttatt ttt gcc att acc agt cag agg cag 1104 Tyr Trp Leu Gly Asn Glu Phe IlePhe Ala Ile Thr Ser Gln Arg Gln 355 360 365 tac atg cta aga att gag ttaatg gac tgg gaa ggg aac cga gcc tat 1152 Tyr Met Leu Arg Ile Glu Leu MetAsp Trp Glu Gly Asn Arg Ala Tyr 370 375 380 tca cag tat gac aga ttc cacata gga aat gaa aag caa aac tat agg 1200 Ser Gln Tyr Asp Arg Phe His IleGly Asn Glu Lys Gln Asn Tyr Arg 385 390 395 400 ttg tat tta aaa ggt cacact ggg aca gca gga aaa cag agc agc ctg 1248 Leu Tyr Leu Lys Gly His ThrGly Thr Ala Gly Lys Gln Ser Ser Leu 405 410 415 atc tta cac ggt gct gatttc agc act aaa gat gct gat aat gac aac 1296 Ile Leu His Gly Ala Asp PheSer Thr Lys Asp Ala Asp Asn Asp Asn 420 425 430 tgt atg tgc aaa tgt gccctc atg tta aca gga gga tgg tgg ttt gat 1344 Cys Met Cys Lys Cys Ala LeuMet Leu Thr Gly Gly Trp Trp Phe Asp 435 440 445 gct tgt ggc ccc tcc aatcta aat gga atg ttc tat act gcg gga caa 1392 Ala Cys Gly Pro Ser Asn LeuAsn Gly Met Phe Tyr Thr Ala Gly Gln 450 455 460 aac cat gga aaa ctg aatggg ata aag tgg cac tac ttc aaa ggg ccc 1440 Asn His Gly Lys Leu Asn GlyIle Lys Trp His Tyr Phe Lys Gly Pro 465 470 475 480 agt tac tcc tta cgttcc aca act atg atg att cga cct tta gat ttt 1488 Ser Tyr Ser Leu Arg SerThr Thr Met Met Ile Arg Pro Leu Asp Phe 485 490 495 tga 1491 22 496 PRTArtificial Sequence Description of Artificial Sequence Chimeric 22 MetTrp Gln Ile Val Phe Phe Thr Leu Ser Cys Asp Leu Val Leu Ala 1 5 10 15Ala Ala Tyr Asn Asn Phe Arg Lys Ser Met Asp Ser Ile Gly Lys Lys 20 25 30Gln Tyr Gln Val Gln His Gly Ser Cys Ser Tyr Thr Phe Leu Leu Pro 35 40 45Glu Met Asp Asn Cys Arg Ser Ser Ser Ser Pro Tyr Val Ser Asn Ala 50 55 60Val Gln Arg Asp Ala Pro Leu Glu Tyr Asp Asp Ser Val Gln Arg Leu 65 70 7580 Gln Val Leu Glu Asn Ile Met Glu Asn Asn Thr Gln Trp Leu Met Lys 85 9095 Leu Glu Asn Tyr Ile Gln Asp Asn Met Lys Lys Glu Met Val Glu Ile 100105 110 Gln Gln Asn Ala Val Gln Asn Gln Thr Ala Val Met Ile Glu Ile Gly115 120 125 Thr Asn Leu Leu Asn Gln Thr Ala Glu Gln Thr Arg Lys Leu ThrAsp 130 135 140 Val Glu Ala Gln Val Leu Asn Gln Thr Thr Arg Leu Glu LeuGln Leu 145 150 155 160 Leu Glu His Ser Leu Ser Thr Asn Lys Leu Glu LysGln Ile Leu Asp 165 170 175 Gln Thr Ser Glu Ile Asn Lys Leu Gln Asp LysAsn Ser Phe Leu Glu 180 185 190 Lys Lys Val Leu Ala Met Glu Asp Lys HisIle Ile Gln Leu Gln Ser 195 200 205 Ile Lys Glu Glu Lys Asp Gln Leu GlnVal Leu Val Ser Lys Gln Asn 210 215 220 Ser Ile Ile Glu Glu Leu Glu LysLys Ile Val Thr Ala Thr Val Asn 225 230 235 240 Asn Ser Val Leu Gln LysGln Gln His Asp Leu Met Glu Thr Val Asn 245 250 255 Asn Leu Leu Thr MetMet Ser Thr Ser Asn Ser Ala Lys Asp Pro Thr 260 265 270 Val Ala Lys GluGlu Gln Ile Ser Phe Arg Asp Cys Ala Asp Val Tyr 275 280 285 Gln Ala GlyPhe Asn Lys Ser Gly Ile Tyr Thr Ile Tyr Ile Asn Asn 290 295 300 Met ProGlu Pro Lys Lys Val Phe Cys Asn Met Asp Val Asn Gly Gly 305 310 315 320Gly Trp Thr Val Ile Gln His Arg Glu Asp Gly Ser Leu Asp Phe Gln 325 330335 Arg Gly Trp Lys Glu Tyr Lys Met Gly Phe Gly Asn Pro Ser Gly Glu 340345 350 Tyr Trp Leu Gly Asn Glu Phe Ile Phe Ala Ile Thr Ser Gln Arg Gln355 360 365 Tyr Met Leu Arg Ile Glu Leu Met Asp Trp Glu Gly Asn Arg AlaTyr 370 375 380 Ser Gln Tyr Asp Arg Phe His Ile Gly Asn Glu Lys Gln AsnTyr Arg 385 390 395 400 Leu Tyr Leu Lys Gly His Thr Gly Thr Ala Gly LysGln Ser Ser Leu 405 410 415 Ile Leu His Gly Ala Asp Phe Ser Thr Lys AspAla Asp Asn Asp Asn 420 425 430 Cys Met Cys Lys Cys Ala Leu Met Leu ThrGly Gly Trp Trp Phe Asp 435 440 445 Ala Cys Gly Pro Ser Asn Leu Asn GlyMet Phe Tyr Thr Ala Gly Gln 450 455 460 Asn His Gly Lys Leu Asn Gly IleLys Trp His Tyr Phe Lys Gly Pro 465 470 475 480 Ser Tyr Ser Leu Arg SerThr Thr Met Met Ile Arg Pro Leu Asp Phe 485 490 495 23 1500 DNAArtificial Sequence Description of Artificial Sequence Chimeric 23 atgaca gtt ttc ctt tcc ttt gct ttc ctc gct gcc att ctg act cac 48 Met ThrVal Phe Leu Ser Phe Ala Phe Leu Ala Ala Ile Leu Thr His 1 5 10 15 ataggg tgc agc aat cag cgc cga agt cca gaa aac agt ggg aga aga 96 Ile GlyCys Ser Asn Gln Arg Arg Ser Pro Glu Asn Ser Gly Arg Arg 20 25 30 tat aaccgg att caa cat ggg caa tgt gcc tac act ttc att ctt cca 144 Tyr Asn ArgIle Gln His Gly Gln Cys Ala Tyr Thr Phe Ile Leu Pro 35 40 45 gaa cac gatggc aac tgt cgt gag agt acg aca gac cag tac aac aca 192 Glu His Asp GlyAsn Cys Arg Glu Ser Thr Thr Asp Gln Tyr Asn Thr 50 55 60 aac gct ctg cagaga gat gct cca cac gtg gaa ccg gat gac tcg gtg 240 Asn Ala Leu Gln ArgAsp Ala Pro His Val Glu Pro Asp Asp Ser Val 65 70 75 80 cag agg ctg caagtg ctg gag aac atc atg gaa aac aac act cag tgg 288 Gln Arg Leu Gln ValLeu Glu Asn Ile Met Glu Asn Asn Thr Gln Trp 85 90 95 cta atg aag ctt gagaat tat atc cag gac aac atg aag aaa gaa atg 336 Leu Met Lys Leu Glu AsnTyr Ile Gln Asp Asn Met Lys Lys Glu Met 100 105 110 gta gag ata cag cagaat gca gta cag aac cag acg gct gtg atg ata 384 Val Glu Ile Gln Gln AsnAla Val Gln Asn Gln Thr Ala Val Met Ile 115 120 125 gaa ata ggg aca aacctg ttg aac caa aca gct gag caa acg cgg aag 432 Glu Ile Gly Thr Asn LeuLeu Asn Gln Thr Ala Glu Gln Thr Arg Lys 130 135 140 tta act gat gtg gaagcc caa gta tta aat cag acc acg aga ctt gaa 480 Leu Thr Asp Val Glu AlaGln Val Leu Asn Gln Thr Thr Arg Leu Glu 145 150 155 160 ctt cag ctc ttggaa cac tcc ctc tcg aca aac aaa ttg gaa aaa cag 528 Leu Gln Leu Leu GluHis Ser Leu Ser Thr Asn Lys Leu Glu Lys Gln 165 170 175 att ttg gac cagacc agt gaa ata aac aaa ttg caa gat aag aac agt 576 Ile Leu Asp Gln ThrSer Glu Ile Asn Lys Leu Gln Asp Lys Asn Ser 180 185 190 ttc cta gaa aagaag gtg cta gct atg gaa gac aag cac atc atc caa 624 Phe Leu Glu Lys LysVal Leu Ala Met Glu Asp Lys His Ile Ile Gln 195 200 205 cta cag tca ataaaa gaa gag aaa gat cag cta cag gtg tta gta tcc 672 Leu Gln Ser Ile LysGlu Glu Lys Asp Gln Leu Gln Val Leu Val Ser 210 215 220 aag caa aat tccatc att gaa gaa cta gaa aaa aaa ata gtg act gcc 720 Lys Gln Asn Ser IleIle Glu Glu Leu Glu Lys Lys Ile Val Thr Ala 225 230 235 240 acg gtg aataat tca gtt ctt caa aag cag caa cat gat ctc atg gag 768 Thr Val Asn AsnSer Val Leu Gln Lys Gln Gln His Asp Leu Met Glu 245 250 255 aca gtt aataac tta ctg act atg atg tcc aca tca aac tca gct aag 816 Thr Val Asn AsnLeu Leu Thr Met Met Ser Thr Ser Asn Ser Ala Lys 260 265 270 gac ccc actgtt gct aaa gaa gaa caa atc agc ttc aga gac tgt gct 864 Asp Pro Thr ValAla Lys Glu Glu Gln Ile Ser Phe Arg Asp Cys Ala 275 280 285 gaa gta ttcaaa tca gga cac acc aca aat ggc atc tac acg tta aca 912 Glu Val Phe LysSer Gly His Thr Thr Asn Gly Ile Tyr Thr Leu Thr 290 295 300 ttc cct aattct aca gaa gag atc aag gcc tac tgt gac atg gaa gct 960 Phe Pro Asn SerThr Glu Glu Ile Lys Ala Tyr Cys Asp Met Glu Ala 305 310 315 320 gga ggaggc ggg tgg aca att att cag cga cgt gag gat ggc agc gtt 1008 Gly Gly GlyGly Trp Thr Ile Ile Gln Arg Arg Glu Asp Gly Ser Val 325 330 335 gat tttcag agg act tgg aaa gaa tat aaa gtg gga ttt ggt aac cct 1056 Asp Phe GlnArg Thr Trp Lys Glu Tyr Lys Val Gly Phe Gly Asn Pro 340 345 350 tca ggagaa tat tgg ctg gga aat gag ttt gtt tcg caa ctg act aat 1104 Ser Gly GluTyr Trp Leu Gly Asn Glu Phe Val Ser Gln Leu Thr Asn 355 360 365 cag caacgc tat gtg ctt aaa ata cac ctt aaa gac tgg gaa ggg aat 1152 Gln Gln ArgTyr Val Leu Lys Ile His Leu Lys Asp Trp Glu Gly Asn 370 375 380 gag gcttac tca ttg tat gaa cat ttc tat ctc tca agt gaa gaa ctc 1200 Glu Ala TyrSer Leu Tyr Glu His Phe Tyr Leu Ser Ser Glu Glu Leu 385 390 395 400 aattat agg att cac ctt aaa gga ctt aca ggg aca gcc ggc aaa ata 1248 Asn TyrArg Ile His Leu Lys Gly Leu Thr Gly Thr Ala Gly Lys Ile 405 410 415 agcagc atc agc caa cca gga aat gat ttt agc aca aag gat gga gac 1296 Ser SerIle Ser Gln Pro Gly Asn Asp Phe Ser Thr Lys Asp Gly Asp 420 425 430 aacgac aaa tgt att tgc aaa tgt tca caa atg cta aca gga ggc tgg 1344 Asn AspLys Cys Ile Cys Lys Cys Ser Gln Met Leu Thr Gly Gly Trp 435 440 445 tggttt gat gca tgt ggt cct tcc aac ttg aac gga atg tac tat cca 1392 Trp PheAsp Ala Cys Gly Pro Ser Asn Leu Asn Gly Met Tyr Tyr Pro 450 455 460 cagagg cag aac aca aat aag ttc aac ggc att aaa tgg tac tac tgg 1440 Gln ArgGln Asn Thr Asn Lys Phe Asn Gly Ile Lys Trp Tyr Tyr Trp 465 470 475 480aaa ggc tca ggc tat tcg ctc aag gcc aca acc atg atg atc cga cca 1488 LysGly Ser Gly Tyr Ser Leu Lys Ala Thr Thr Met Met Ile Arg Pro 485 490 495gca gat ttc taa 1500 Ala Asp Phe 24 499 PRT Artificial SequenceDescription of Artificial Sequence Chimeric 24 Met Thr Val Phe Leu SerPhe Ala Phe Leu Ala Ala Ile Leu Thr His 1 5 10 15 Ile Gly Cys Ser AsnGln Arg Arg Ser Pro Glu Asn Ser Gly Arg Arg 20 25 30 Tyr Asn Arg Ile GlnHis Gly Gln Cys Ala Tyr Thr Phe Ile Leu Pro 35 40 45 Glu His Asp Gly AsnCys Arg Glu Ser Thr Thr Asp Gln Tyr Asn Thr 50 55 60 Asn Ala Leu Gln ArgAsp Ala Pro His Val Glu Pro Asp Asp Ser Val 65 70 75 80 Gln Arg Leu GlnVal Leu Glu Asn Ile Met Glu Asn Asn Thr Gln Trp 85 90 95 Leu Met Lys LeuGlu Asn Tyr Ile Gln Asp Asn Met Lys Lys Glu Met 100 105 110 Val Glu IleGln Gln Asn Ala Val Gln Asn Gln Thr Ala Val Met Ile 115 120 125 Glu IleGly Thr Asn Leu Leu Asn Gln Thr Ala Glu Gln Thr Arg Lys 130 135 140 LeuThr Asp Val Glu Ala Gln Val Leu Asn Gln Thr Thr Arg Leu Glu 145 150 155160 Leu Gln Leu Leu Glu His Ser Leu Ser Thr Asn Lys Leu Glu Lys Gln 165170 175 Ile Leu Asp Gln Thr Ser Glu Ile Asn Lys Leu Gln Asp Lys Asn Ser180 185 190 Phe Leu Glu Lys Lys Val Leu Ala Met Glu Asp Lys His Ile IleGln 195 200 205 Leu Gln Ser Ile Lys Glu Glu Lys Asp Gln Leu Gln Val LeuVal Ser 210 215 220 Lys Gln Asn Ser Ile Ile Glu Glu Leu Glu Lys Lys IleVal Thr Ala 225 230 235 240 Thr Val Asn Asn Ser Val Leu Gln Lys Gln GlnHis Asp Leu Met Glu 245 250 255 Thr Val Asn Asn Leu Leu Thr Met Met SerThr Ser Asn Ser Ala Lys 260 265 270 Asp Pro Thr Val Ala Lys Glu Glu GlnIle Ser Phe Arg Asp Cys Ala 275 280 285 Glu Val Phe Lys Ser Gly His ThrThr Asn Gly Ile Tyr Thr Leu Thr 290 295 300 Phe Pro Asn Ser Thr Glu GluIle Lys Ala Tyr Cys Asp Met Glu Ala 305 310 315 320 Gly Gly Gly Gly TrpThr Ile Ile Gln Arg Arg Glu Asp Gly Ser Val 325 330 335 Asp Phe Gln ArgThr Trp Lys Glu Tyr Lys Val Gly Phe Gly Asn Pro 340 345 350 Ser Gly GluTyr Trp Leu Gly Asn Glu Phe Val Ser Gln Leu Thr Asn 355 360 365 Gln GlnArg Tyr Val Leu Lys Ile His Leu Lys Asp Trp Glu Gly Asn 370 375 380 GluAla Tyr Ser Leu Tyr Glu His Phe Tyr Leu Ser Ser Glu Glu Leu 385 390 395400 Asn Tyr Arg Ile His Leu Lys Gly Leu Thr Gly Thr Ala Gly Lys Ile 405410 415 Ser Ser Ile Ser Gln Pro Gly Asn Asp Phe Ser Thr Lys Asp Gly Asp420 425 430 Asn Asp Lys Cys Ile Cys Lys Cys Ser Gln Met Leu Thr Gly GlyTrp 435 440 445 Trp Phe Asp Ala Cys Gly Pro Ser Asn Leu Asn Gly Met TyrTyr Pro 450 455 460 Gln Arg Gln Asn Thr Asn Lys Phe Asn Gly Ile Lys TrpTyr Tyr Trp 465 470 475 480 Lys Gly Ser Gly Tyr Ser Leu Lys Ala Thr ThrMet Met Ile Arg Pro 485 490 495 Ala Asp Phe 25 1488 DNA ArtificialSequence Description of Artificial Sequence Chimeric 25 atg tgg cag attgtt ttc ttt act ctg agc tgt gat ctt gtc ttg gcc 48 Met Trp Gln Ile ValPhe Phe Thr Leu Ser Cys Asp Leu Val Leu Ala 1 5 10 15 gca gcc tat aacaac ttt cgg aag agc atg gac agc ata gga aag aag 96 Ala Ala Tyr Asn AsnPhe Arg Lys Ser Met Asp Ser Ile Gly Lys Lys 20 25 30 caa tat cag gtc cagcat ggg tcc tgc agc tac act ttc ctc ctg cca 144 Gln Tyr Gln Val Gln HisGly Ser Cys Ser Tyr Thr Phe Leu Leu Pro 35 40 45 gag atg gac aac tgc cgctct tcc tcc agc ccc tac gtg tcc aat gct 192 Glu Met Asp Asn Cys Arg SerSer Ser Ser Pro Tyr Val Ser Asn Ala 50 55 60 gtg cag agg gac gcg ccg ctcgaa tac gat ttc tct tcc cag aaa ctt 240 Val Gln Arg Asp Ala Pro Leu GluTyr Asp Phe Ser Ser Gln Lys Leu 65 70 75 80 caa cat ctg gaa cat gtg atggaa aat tat act cag tgg ctg caa aaa 288 Gln His Leu Glu His Val Met GluAsn Tyr Thr Gln Trp Leu Gln Lys 85 90 95 ctt gag aat tac att gtg gaa aacatg aag tcg gag atg gcc cag ata 336 Leu Glu Asn Tyr Ile Val Glu Asn MetLys Ser Glu Met Ala Gln Ile 100 105 110 cag cag aat gca gtt cag aac cacacg gct acc atg ctg gag ata gga 384 Gln Gln Asn Ala Val Gln Asn His ThrAla Thr Met Leu Glu Ile Gly 115 120 125 acc agc ctc ctc tct cag act gcagag cag acc aga aag ctg aca gat 432 Thr Ser Leu Leu Ser Gln Thr Ala GluGln Thr Arg Lys Leu Thr Asp 130 135 140 gtt gag acc cag gta cta aat caaact tct cga ctt gag ata cag ctg 480 Val Glu Thr Gln Val Leu Asn Gln ThrSer Arg Leu Glu Ile Gln Leu 145 150 155 160 ctg gag aat tca tta tcc acctac aag cta gag aag caa ctt ctt caa 528 Leu Glu Asn Ser Leu Ser Thr TyrLys Leu Glu Lys Gln Leu Leu Gln 165 170 175 cag aca aat gaa atc ttg aagatc cat gaa aaa aac agt tta tta gaa 576 Gln Thr Asn Glu Ile Leu Lys IleHis Glu Lys Asn Ser Leu Leu Glu 180 185 190 cat aaa atc tta gaa atg gaagga aaa cac aag gaa gag ttg gac acc 624 His Lys Ile Leu Glu Met Glu GlyLys His Lys Glu Glu Leu Asp Thr 195 200 205 tta aag gaa gag aaa gag aacctt caa ggc ttg gtt act cgt caa aca 672 Leu Lys Glu Glu Lys Glu Asn LeuGln Gly Leu Val Thr Arg Gln Thr 210 215 220 tat ata atc cag gag ctg gaaaag caa tta aac aga gct acc acc aac 720 Tyr Ile Ile Gln Glu Leu Glu LysGln Leu Asn Arg Ala Thr Thr Asn 225 230 235 240 aac agt gtc ctt cag aagcag caa ctg gag ctg atg gac aca gtc cac 768 Asn Ser Val Leu Gln Lys GlnGln Leu Glu Leu Met Asp Thr Val His 245 250 255 aac ctt gtc aat ctt tgcact aaa gaa ggt gtt tta cta aag gga gga 816 Asn Leu Val Asn Leu Cys ThrLys Glu Gly Val Leu Leu Lys Gly Gly 260 265 270 aaa aga gag gaa gag aaacca ttt aga gac tgt gca gat gta tat caa 864 Lys Arg Glu Glu Glu Lys ProPhe Arg Asp Cys Ala Asp Val Tyr Gln 275 280 285 gct ggt ttt aat aaa agtgga atc tac act att tat att aat aat atg 912 Ala Gly Phe Asn Lys Ser GlyIle Tyr Thr Ile Tyr Ile Asn Asn Met 290 295 300 cca gaa ccc aaa aag gtgttt tgc aat atg gat gtc aat ggg gga ggt 960 Pro Glu Pro Lys Lys Val PheCys Asn Met Asp Val Asn Gly Gly Gly 305 310 315 320 tgg act gta ata caacat cgt gaa gat gga agt cta gat ttc caa aga 1008 Trp Thr Val Ile Gln HisArg Glu Asp Gly Ser Leu Asp Phe Gln Arg 325 330 335 ggc tgg aag gaa tataaa atg ggt ttt gga aat ccc tcc ggt gaa tat 1056 Gly Trp Lys Glu Tyr LysMet Gly Phe Gly Asn Pro Ser Gly Glu Tyr 340 345 350 tgg ctg ggg aat gagttt att ttt gcc att acc agt cag agg cag tac 1104 Trp Leu Gly Asn Glu PheIle Phe Ala Ile Thr Ser Gln Arg Gln Tyr 355 360 365 atg cta aga att gagtta atg gac tgg gaa ggg aac cga gcc tat tca 1152 Met Leu Arg Ile Glu LeuMet Asp Trp Glu Gly Asn Arg Ala Tyr Ser 370 375 380 cag tat gac aga ttccac ata gga aat gaa aag caa aac tat agg ttg 1200 Gln Tyr Asp Arg Phe HisIle Gly Asn Glu Lys Gln Asn Tyr Arg Leu 385 390 395 400 tat tta aaa ggtcac act ggg aca gca gga aaa cag agc agc ctg atc 1248 Tyr Leu Lys Gly HisThr Gly Thr Ala Gly Lys Gln Ser Ser Leu Ile 405 410 415 tta cac ggt gctgat ttc agc act aaa gat gct gat aat gac aac tgt 1296 Leu His Gly Ala AspPhe Ser Thr Lys Asp Ala Asp Asn Asp Asn Cys 420 425 430 atg tgc aaa tgtgcc ctc atg tta aca gga gga tgg tgg ttt gat gct 1344 Met Cys Lys Cys AlaLeu Met Leu Thr Gly Gly Trp Trp Phe Asp Ala 435 440 445 tgt ggc ccc tccaat cta aat gga atg ttc tat act gcg gga caa aac 1392 Cys Gly Pro Ser AsnLeu Asn Gly Met Phe Tyr Thr Ala Gly Gln Asn 450 455 460 cat gga aaa ctgaat ggg ata aag tgg cac tac ttc aaa ggg ccc agt 1440 His Gly Lys Leu AsnGly Ile Lys Trp His Tyr Phe Lys Gly Pro Ser 465 470 475 480 tac tcc ttacgt tcc aca act atg atg att cga cct tta gat ttt tga 1488 Tyr Ser Leu ArgSer Thr Thr Met Met Ile Arg Pro Leu Asp Phe 485 490 495 26 495 PRTArtificial Sequence Description of Artificial Sequence Chimeric 26 MetTrp Gln Ile Val Phe Phe Thr Leu Ser Cys Asp Leu Val Leu Ala 1 5 10 15Ala Ala Tyr Asn Asn Phe Arg Lys Ser Met Asp Ser Ile Gly Lys Lys 20 25 30Gln Tyr Gln Val Gln His Gly Ser Cys Ser Tyr Thr Phe Leu Leu Pro 35 40 45Glu Met Asp Asn Cys Arg Ser Ser Ser Ser Pro Tyr Val Ser Asn Ala 50 55 60Val Gln Arg Asp Ala Pro Leu Glu Tyr Asp Phe Ser Ser Gln Lys Leu 65 70 7580 Gln His Leu Glu His Val Met Glu Asn Tyr Thr Gln Trp Leu Gln Lys 85 9095 Leu Glu Asn Tyr Ile Val Glu Asn Met Lys Ser Glu Met Ala Gln Ile 100105 110 Gln Gln Asn Ala Val Gln Asn His Thr Ala Thr Met Leu Glu Ile Gly115 120 125 Thr Ser Leu Leu Ser Gln Thr Ala Glu Gln Thr Arg Lys Leu ThrAsp 130 135 140 Val Glu Thr Gln Val Leu Asn Gln Thr Ser Arg Leu Glu IleGln Leu 145 150 155 160 Leu Glu Asn Ser Leu Ser Thr Tyr Lys Leu Glu LysGln Leu Leu Gln 165 170 175 Gln Thr Asn Glu Ile Leu Lys Ile His Glu LysAsn Ser Leu Leu Glu 180 185 190 His Lys Ile Leu Glu Met Glu Gly Lys HisLys Glu Glu Leu Asp Thr 195 200 205 Leu Lys Glu Glu Lys Glu Asn Leu GlnGly Leu Val Thr Arg Gln Thr 210 215 220 Tyr Ile Ile Gln Glu Leu Glu LysGln Leu Asn Arg Ala Thr Thr Asn 225 230 235 240 Asn Ser Val Leu Gln LysGln Gln Leu Glu Leu Met Asp Thr Val His 245 250 255 Asn Leu Val Asn LeuCys Thr Lys Glu Gly Val Leu Leu Lys Gly Gly 260 265 270 Lys Arg Glu GluGlu Lys Pro Phe Arg Asp Cys Ala Asp Val Tyr Gln 275 280 285 Ala Gly PheAsn Lys Ser Gly Ile Tyr Thr Ile Tyr Ile Asn Asn Met 290 295 300 Pro GluPro Lys Lys Val Phe Cys Asn Met Asp Val Asn Gly Gly Gly 305 310 315 320Trp Thr Val Ile Gln His Arg Glu Asp Gly Ser Leu Asp Phe Gln Arg 325 330335 Gly Trp Lys Glu Tyr Lys Met Gly Phe Gly Asn Pro Ser Gly Glu Tyr 340345 350 Trp Leu Gly Asn Glu Phe Ile Phe Ala Ile Thr Ser Gln Arg Gln Tyr355 360 365 Met Leu Arg Ile Glu Leu Met Asp Trp Glu Gly Asn Arg Ala TyrSer 370 375 380 Gln Tyr Asp Arg Phe His Ile Gly Asn Glu Lys Gln Asn TyrArg Leu 385 390 395 400 Tyr Leu Lys Gly His Thr Gly Thr Ala Gly Lys GlnSer Ser Leu Ile 405 410 415 Leu His Gly Ala Asp Phe Ser Thr Lys Asp AlaAsp Asn Asp Asn Cys 420 425 430 Met Cys Lys Cys Ala Leu Met Leu Thr GlyGly Trp Trp Phe Asp Ala 435 440 445 Cys Gly Pro Ser Asn Leu Asn Gly MetPhe Tyr Thr Ala Gly Gln Asn 450 455 460 His Gly Lys Leu Asn Gly Ile LysTrp His Tyr Phe Lys Gly Pro Ser 465 470 475 480 Tyr Ser Leu Arg Ser ThrThr Met Met Ile Arg Pro Leu Asp Phe 485 490 495 27 47 DNA ArtificialSequence Description of Artificial Sequence PCR Primer 27 gcatgctatctcgagccacc atgctctccc agctagccat gctgcag 47 28 55 DNA ArtificialSequence Description of Artificial Sequence PCR Primer 28 gtgtcgacgcggccgctcta gatcagactt agatgtccaa aggccgtatc atcat 55 29 25 DNAArtificial Sequence Description of Artificial Sequence PCR Primer 29cctctgggctcgccagtttgttagg 25 30 19 DNA Artificial Sequence Descriptionof Artificial Sequence PCR Primer 30 ccagctggcagatatcagg 19

What is claimed is:
 1. An isolated nucleic acid molecule encoding afusion protein, wherein said fusion protein comprises a modified TIE-2ligand 2 protein and human immunoglobulin gamma-1 constant region (IgG1Fc), wherein TIE-2 ligand 2 comprises an N-terminal domain, acoiled-coil domain, and C-terminal fibrinogen-like domain, and themodified TIE-2 ligand protein has the N-terminal and coiled-coil domainsdeleted and the fibrinogen-like domain comprising amino acids 281-496 ofSEQ ID NO:
 6. 2. A vector comprising the nucleic acid molecule ofclaim
 1. 3. The vector of claim 2, operatively linked to an expressioncontrol sequence capable of directing its expression in a host cell. 4.The vector of claim 3, which is a plasmid.
 5. An isolated host-vectorsystem for the production of a modified TIE-2 ligand 2, comprising thevector of claim 2 in a host cell.
 6. The isolated host-vector system ofclaim 5, wherein the host cell is a bacterial, yeast, insect, ormammalian cell.
 7. A method for producing a modified TIE-2 ligand 2protein, comprising growing the isolated host-vector system of claim 6under conditions permitting production of a modified TIE2-ligand 2protein, and recovering the polypeptide so produced.